Table of Contents - Michigan State Universitycore.phmtox.msu.edu/Scheduling/ItemDocs/20/Odyssey... · LI-COR Biosciences Doc# 988-09288 Page 2 II. Western Detection Methods Nitrocellulose

Table of ContentsProtocol Publication Number

Western Blot Analysis ................................................................................................................................................................988-09288

In-Gel Western Detection ...........................................................................................................................................................988-08329

Northern Blot Analysis Using Biotin-Labeled Probes ..............................................................................................................988-09394

Southern Blot Analysis Using Biotin PCR Labeled Probes ......................................................................................................988-09395

In-Cell Western Analysis: For Assessing Response of A431 Cells to Stimulation with Epidermal

Growth Factor .....................................................................................................................................................................988-08332

In-Cell Western Assay: IRDye® 800CW EGF Competition and Binding Assay Using A431 Cells .........................................988-08625

In-Cell Western Assay: Complete Sample Protocol for Measuring IC50 of Inhibitor PD168393 in

A431 Cells Responding to Epidermal Growth Factor ......................................................................................................988-08599

In-Cell Western Assay: Complete Sample Protocol for Measuring IC50 of Inhibitor U0126 in NIH3T3

Responding to Acidic Fibroblast Growth Factor (aFGF-1) ...............................................................................................988-08334

In-Cell Western Assay: Complete Sample Protocol Detailing the Seeding, Stimulation, and

Detection of the HeLa Cellular Response to Epidermal Growth Factor .........................................................................988-08335


Detection of the NIH3T3 Cellular Response to Platelet Derived Growth Factor BB (PDGF-BB) ...................................988-08336


Detection of the NIH3T3 Cellular Response to Acidic Fibroblast Growth Factor (aFGF-1) ...........................................988-08337

In-Cell Western Assay: Complete Apoptosis Assay Example Detailing the Seeding, Induction, and

Detection of the HeLa Cellular Response to Anisomycin Treatment ..............................................................................988-08338

In-Cell Western Assay: Phospho-p53 Detection in COS Cells in Response to Hydroxyurea ................................................988-08339

In-Cell Western Assay: Phospho-p38 Detection in HeLa Cells in Response to Anisomycin .................................................988-09368

In-Cell Western Assay: Complete Sample Protocol for PMA-induced ERK Activation in Suspension Cell Lines ...............988-08340

Technical Note: FAQs for Suspension Cells for ICW Protocols ...............................................................................................988-08341

In-Cell Western™ Assay Kit I and Kit II Pack Insert ................................................................................................................... 988-08911

Electrophoretic Mobility Shift Assay (EMSA) Using IRDye®-Labeled DNA ............................................................................ 982-07487

GEArray® Q Series Gene Expression Arrays ............................................................................................................................988-08344

Syto® 60 Staining of Nucleic Acids on Gels ..............................................................................................................................988-08345

Active Motif TransAM™ Transcription Factor Assays on the Odyssey System ......................................................................988-08346

Adapting TranSignal™ Target Gene Arrays for the Odyssey System .....................................................................................988-08347

Cartesian Array™ Human Cytokine Set 1 (Biosource International, Camarillo, CA) on the Odyssey System ....................988-08348

RayBio® Cytokine Antibody Arrays (RayBiotech Inc., Norcross, GA) on the Odyssey System .............................................988-08349

Cayman Chemical PPARγ Transcription Factor Arrays on the Odyssey System ....................................................................988-09402

TranSignal™ Cytokine Antibody Arrays (Panomics Inc., Redwood City, CA) on the Odyssey System ...............................988-08350

Fluorophore-Linked Immunosorbant Assay (FLISA) Recommendations ..............................................................................988-08352

Tips for Antibody and Lysate Arraying ..................................................................................................................................... 988-08014

Scanning a Mouse on Odyssey: Hints and Tips .......................................................................................................................988-08080

IRDye® 800CW Protein Labeling Kit - High MW .......................................................................................................................988-08616

IRDye® 800CW Protein Labeling Kit - Low MW ........................................................................................................................988-08617

IRDye® 800CW Protein Labeling Kit - Microscale .....................................................................................................................988-08618

LI-COR MAKES NO WARRANTY OF ANY KIND WITH REGARD TO THIS MATERIAL, INCLUDING, BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE. LI-COR shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance, or use of this material. © 2004-2008 LI-COR, inc. This document contains proprietary information which is protected by copyright. All rights are reserved. No part of this document may be photocopied, reproduced, or translated to another language without prior written consent of LI-COR, Inc. The information contained in this document is subject to change without notice. Publication Number 984-09546. LI-COR is an ISO 9001 registered company. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, inc. The Odyssey Infrared Imager and IRDyes are covered by U.S. patents and patents pending.

988-09547

Odyssey

Infrared Imaging System

®

Western Blot Analysis

Doc# 988-09288

Published January 2003. Revised October, 2007. The most recent ver-sion of this protocol is posted at http://biosupport.licor.com/support

®

Odyssey

®


Doc# 988-09288Page 1 www.licor.com

Contents

Page

I. Required Reagents...........................................................................................1

II. Western Detection Methods .............................................................................2

III. Guidelines for Two-Color Detection...................................................................4

IV. Stripping the Membrane ...................................................................................5

V. Adapting Western Blotting Protocols for Odyssey Detection ............................5

VI. General Tips .....................................................................................................6

VII. Imaging of Coomassie

®

-Stained Protein Gels..................................................7

VIII. Troubleshooting Guide......................................................................................8

I. Required Reagents

• Blotted nitrocellulose (LI-COR, Cat. #926-31090) or PVDF membrane • Odyssey

®

Blocking Buffer (LI-COR, Cat. #927-40000)• Primary antibodies• Infrared IRDye

®

-labeled secondary antibodies (LI-COR)*• Tween

®

-20• PBS wash buffer (LI-COR, Cat. #928-40018, or 928-40020)• Double distilled water• Methanol for wetting of PVDF• SDS (if desired)• Other blocking buffers (if desired)

• NewBlot™ Stripping Buffer, if desired, for nitrocellulose (LI-COR, Cat. #928-40030) or PVDF (LI-COR, Cat. # 928-40032) membranes

* IRDye 800CW-labeled secondary antibodies are also available from Rockland Immunochemicals, Inc. Alexa Fluor

®

680-labeled secondary anti-bodies are available from Invitrogen Corporation.

Fluorescent Dyes Appropriate for Use with the Odyssey System

Dye Sensitivity Odyssey Channel

IRDye

®

800CW + + + 800IRDye

®

800 + + + 800IRDye

®

680 + + + 700IRDye

®

700DX + + 700Alexa Fluor

®

680 + + + 700Alexa Fluor

®

700 + + 700Alexa Fluor

®

750 + + 700/800

(not recommended; signal appears in both channels)

Alexa Fluor

®

647 + 700

Cy

®

5.5 + + 700

Cy

®

5 + 700

The most current informa-tion on dye compatibility can be found on the LI-COR

web site.

LI-COR Biosciences

Doc# 988-09288 Page 2www.licor.com

II. Western Detection Methods

Nitrocellulose or PVDF membranes may be used for protein blotting, but nitrocellulose membrane is rec-ommended for maximum performance. Pure cast nitrocellulose is generally preferable to supported nitro-cellulose. Protein should be transferred from gel to membrane by standard procedures. Membranes should be handled only by their edges, with clean forceps.

After transfer, perform the following steps:

1. Wet the membrane in PBS for several minutes. If using a PVDF membrane that has been allowed to dry, pre-wet briefly in 100% methanol and rinse with double distilled water before incubating in PBS.

Notes:

• Ink from most pens and markers will fluoresce on Odyssey. The ink may wash off and re-deposit elsewhere on the membrane, creating blotches and streaks. Mark with pencil or the provided Odyssey pen to avoid this problem. Use pencil for PVDF membrane (wetting in methanol will cause ink to run).

2. Block the membrane in Odyssey Blocking Buffer for 1 hour. Be sure to use sufficient blocking buffer to cover the membrane (a minimum of 0.4 ml/cm

2

is suggested).

Notes:

• Membranes can be blocked overnight at 4°C if desired.

• Odyssey Blocking Buffer often yields higher and more consistent sensitivity and performance than other blockers. Nonfat dry milk or casein dissolved in PBS can also be used for blocking and antibody dilution, but be aware that milk may cause higher background on PVDF membranes. If using casein, a 0.1% solution in 0.2 X PBS buffer is recommended (Hammersten-grade casein is not required).

• Milk-based reagents can interfere with detection when using anti-goat antibodies. They also deteriorate rapidly at 4°C, so diluted antibodies cannot be kept and re-used for more than a few days.

• Odyssey Blocking Buffer can often be diluted at least 1:1 in PBS without loss of performance.

• Odyssey Blocking Buffer can be saved and re-used.

• Blocking solutions containing BSA can be used, but in some cases they may cause high membrane back-ground.

BSA-containing blockers are not generally recommended

and should be used only when the pri-mary antibody requires BSA as blocker.

3. Dilute primary antibody in Odyssey Blocking Buffer. Optimum dilution depends on your antibody and should be determined empirically. A suggested starting range is 1:1000 to 1:5000. To lower background, add 0.1 - 0.2% Tween-20 to the diluted antibody before incubation. The optimum Tween-20 concentration will depend on your antibody.

Notes:

• Two-color detection requires primary antibodies raised in different host species, such as rabbit and mouse. For details, see

III. Guidelines for Two Color Western Detection

.

4. Incubate blot in primary antibody for 60 minutes or longer at room temperature with gentle shaking. Optimum incubation times vary for different primary antibodies. Use enough antibody solution to completely cover the membrane.

5. Wash membrane 4 times for 5 minutes each at room temperature in PBS + 0.1% Tween-20 with gentle shaking, using a generous amount of buffer.

Odyssey

®



Molecular Weight Marker

If you loaded the Odyssey Prestained Molecular Weight Marker (LI-COR, Cat. #928-40000) on your gel before transfer, it will be visible in the 700 nm channel image and also faintly visible in the 800 nm chan-nel image. If the marker is subjected to numerous freeze/thaw cycles, it may degrade. This is observed as multiple, high-molecular weight bands appearing in the 800 nm channel. If this occurs, discard the aliquot and use a fresh one.

Prestained blue molecular weight markers from other sources can also be used with Odyssey. Load 1/3 to 1/5 of the amount you would normally use for Western transfer. Too much marker can result in very strong marker bands that may interfere with visualization of sample lanes. If using multicolored markers, some bands may not be visualized with Odyssey.

6. Dilute the fluorescently-labeled secondary antibody in Odyssey Blocking Buffer. Avoid prolonged exposure of antibody vial to light. Recommended dilution is 1:15,000 (suggested dilution range is 1:5000 to 1:25,000). Add Tween-20 to the diluted antibody as you did for the primary antibody. Add SDS if desired, see

Notes

below.

Notes:

• For detection of small amounts of protein, try using more secondary antibody (1:5000-1:10,000).

• Be careful not to introduce contamination into the antibody vial.

• Diluted secondary antibody can be saved and re-used. Store at 4°C and protect from light. However, for best sensitivity and performance, use freshly diluted antibody solution.

• Adding

0.01% - 0.02% SDS

to the diluted secondary antibody (in addition to Tween-20) will substantially reduce membrane background, particularly when using PVDF. However, DO NOT add SDS during block-ing or to the diluted primary antibody. See

V. Adapting Western Blotting Protocols for Odyssey Detection

for more information about how and why to use SDS in the secondary antibody incubation.

7. Incubate blot in secondary antibody for 30-60 minutes at room temperature with gentle shaking. Protect from light during incubation.

Notes:

• Allowing incubation to proceed more than 60 minutes may increase background.

8. Wash membrane 4 times for 5 minutes each at room temperature in PBS + 0.1% Tween-20 with gentle shaking. Protect from light.

9. Rinse membrane with PBS to remove residual Tween-20. The membrane is now ready to scan.

Notes:

• Scan in the appropriate channels (see


for details).

• Protect the membrane from light until it has been scanned.

• Keep the membrane wet if you plan to strip and re-use it. Once a membrane has dried, stripping is ineffec-tive.

• Blots can be allowed to dry before scanning if desired. Signal strength may be enhanced on a dry mem-brane. The membrane can also be re-wetted for scanning.

• The fluorescent signal on the membrane will remain stable for several months, or longer, if protected from light. Membranes may be stored dry or in PBS buffer at 4°C.

• If signal on membrane is too strong or too weak, re-scan the membrane at a lower or higher scan intensity setting, respectively.

LI-COR Biosciences


Optimization Tips

■

Follow the protocol carefully.

■

No single blocking reagent will be optimal for every antigen-antibody pair. Some primary antibodies may exhibit greatly reduced signal or different nonspecific banding in different blocking solutions. If you have difficulty detecting your target protein, changing the blocking solution may dramatically improve performance. If the primary antibody has worked well in the past using chemiluminescent detection, try that blocking solution for Odyssey detection.

■

Addition of detergent such as Tween-20 can reduce membrane background and non-specific banding. Refer to


for details.

■

To avoid background speckles on blots, use high-quality double distilled water for buffers and rinse plastic dishes well before and after use. Never perform Western incubations or washes in dishes that have been used for Coomassie staining.

■

Membranes should be handled only by their edges, with forceps.

■

After you handle membranes that have been incubating in antibody solutions, clean forceps thoroughly with distilled water and/or ethanol. If forceps are not cleaned after being dipped in antibody solutions, they can cause spots or streaks of background on the membrane that are difficult to wash away.

■

When scanning, always clean the Odyssey scanning surface first to remove dust, residue, and smudges that may affect image quality or contaminate the membrane. If using a silicone mat over your membranes, carefully clean the surface of the mat that will touch the membrane; a dirty mat can deposit dust and residue. Avoid rubbing or wiping the mat with tissue, as this creates more lint and leads to speckling.

■

Do not wrap the membrane in plastic when scanning.

■

If you plan to strip a Western blot, do not allow the membrane to dry. Stripping is ineffective once a membrane has dried, or even partially dried.

III. Guidelines for Two-Color Detection

Two different antigens can be detected simultaneously on the same blot using antibodies labeled with IR dyes that are visualized in different fluorescence channels (700 and 800 nm). Two-color detection requires careful selection of primary and secondary antibodies.

The following guidelines will help you design two color experiments:

■

The two primary antibodies must be derived from different host species

so they can be discriminated by secondary antibodies of different specificities (for example, primaries from rabbit and mouse will be discriminated by anti-rabbit and anti-mouse secondary antibodies).

■

Before combining primary antibodies in a two-color experiment, always perform preliminary blots with each primary antibody alone to determine the expected banding pattern and possible background bands. Slight cross-reactivity may occur and can complicate interpretation of your blot, particularly if the antigen is very abundant. If cross-reactivity is a problem, load less protein or reduce the amount of antibody.

■

One secondary antibody must be labeled with a 700 channel dye, and the other with an 800 channel dye. For a list of fluorescent dyes and the Odyssey channels where they can be visualized, see


.

■

Always use highly cross-adsorbed secondary antibodies for two color detection. Failure to use cross-adsorbed antibodies may result in cross-reactivity.

Odyssey

®



■

For best results, avoid using primary antibodies from mouse and rat together for a two-color experiment. It is not possible to completely adsorb away cross-reactivity because the species are so closely related. If using mouse and rat together, it is crucial to run single-color blots first with each individual antibody to be certain of expected band sizes.

■

If possible, the two secondary antibodies should be derived from the same host species (for example, goat anti-mouse and goat anti-rabbit) to eliminate the chance of the secondaries reacting against one another. The secondary antibodies should not recognize immunoglobulins from other species that may be present in the sample.

When performing a two-color blot, use the standard Western blot protocol with the following modifications:

• Combine the two primary antibodies in the diluted antibody solution in step 3. Incubate simultaneously with membrane (step 4). The primary antibodies

must

be from two different host species.

• Combine the two dye-labeled secondary antibodies in the diluted antibody solution in step 6. Incubate simultaneously with membrane (step 7).

IV. Stripping the Membrane

Typically, both PVDF and nitrocellulose membranes can be stripped up to three times. LI-COR NewBlot™ Stripping Buffer is available under Cat. #928-40030 for nitrocellulose or 928-40032 for PVDF. If a blot is to be stripped, DO NOT allow it to dry before, during, or after imaging (keep the blot as wet as possible). Com-plete usage instructions are given in the NewBlot™ Stripping Buffer pack insert that is shipped with the prod-uct. Before proceeding read the instructions in the pack insert, including the frequently asked questions.


When adapting your Western blotting protocols for Odyssey detection or using a new primary antibody, it is important that you determine the optimal antibody concentrations. Optimization allows you to achieve maximum sensitivity and consistency.

Three parameters should be optimized: primary antibody concentration, dye-labeled secondary antibody concentration, and detergent concentration in the diluted antibodies.

Primary Antibody Concentration

Primary antibodies vary widely in quality, affinity, and concentration. The correct working range for anti-body dilution depends on the characteristics of your primary antibody and the amount of target antigen you want to detect. Suggested dilutions are 1:500, 1:1500, 1:5000, and 1:10,000 (start with the dilution factor you would normally use for chemiluminescent detection). Optimize your primary dilution to achieve max-imum performance and conserve antibody.

Secondary Antibody Concentration

Optimal dilutions of dye-conjugated secondary antibodies should also be determined. Suggested starting dilutions to test are 1:5000, 1:10,000, and 1:20,000. The amount of secondary required varies depending

LI-COR Biosciences


on how much antigen is being detected – abundant proteins with strong signals require less secondary antibody.

Detergent Concentration

Addition of detergents to diluted antibodies can significantly reduce background on the blot. Optimal detergent concentration will vary, depending on the antibodies, membrane type, and blocker used. Keep in mind that some primaries do not bind as tightly as others and may be washed away by too much detergent. Never expose the membrane to detergent until blocking is complete, as this may cause high membrane background.

Tween-20:

• Add Tween-20 to both the primary antibody and secondary antibody solutions when the antibodies are diluted in blocking buffer. A final concentration of 0.1 - 0.2% is recommended for nitrocellulose mem-branes, and a final concentration of 0.1% is recommended for PVDF membranes (higher concentrations of Tween-20 may actually cause increased background on PVDF).

• Wash solutions should contain 0.1% Tween-20.

SDS:

• Adding

0.01 - 0.02% SDS

to the diluted secondary antibody can dramatically reduce overall membrane background and also reduce or eliminate non-specific banding. It is critical to use only a very small amount, because SDS is an ionic detergent and can disrupt antigen-antibody interactions if too much is present at any time during the detection process.

• Addition of SDS is particularly helpful for reducing the higher overall background that is seen with PVDF membrane.

•

DO NOT add SDS during the blocking step or to the diluted primary antibody.

Presence of SDS during binding of the primary antibody to its antigen may greatly reduce signal.

Add SDS only to the diluted secondary antibody.

• When diluting the dye-labeled secondary antibody in blocking buffer, add

both

0.1 - 0.2% Tween-20 and 0.01 - 0.02% SDS to the antibody solution.

• Wash solutions should contain 0.1% Tween-20, but no SDS.

• Some antibody-antigen pairs may be more sensitive to the presence of SDS and may require even lower concentrations of this detergent (less than 0.01%) for best performance. Titrate the amount of SDS to find the best level for the antibodies used.

• If high background is seen when using BSA-containing blocking buffers, adding SDS to the secondary antibody may alleviate the problem.

VI. General Tips

■

Milk-based blockers may contain IgG that can cross-react with anti-goat antibodies. This can significantly increase background and reduce antibody titer. Milk may also contain endogenous biotin or phospho-epitopes that can cause higher background.

■

To stretch the Odyssey Blocking Buffer: recycle your blocking buffer for antibody dilution; dilute 1:1 in PBS; save excess used blocking buffer at 4°C for several days for re-use (use a separate container – do not return to the main bottle).

Odyssey

®



■

Store the antibody vial at 4°C in the dark. Do not thaw and refreeze the vial, as this will affect antibody performance. Minimize exposure to light and take care not to introduce contamination into the vial. Dilute immediately prior to use. If particulates are seen in the antibody solution, centrifuge before use.

■

Protect membrane from light during secondary antibody incubations and washes.

■

Use the narrowest well size possible for your loading volume to concentrate the target protein.

■

The best transfer conditions, membrane, and blocking agent for your experiments will vary, depending on the antigen and antibody. If you have problems with high back ground or low signal level, a good first step is to try a different blocking solution.

■

For maximum sensitivity, use nitrocellulose membrane for transfer.

■

Small amounts of purified protein may not transfer well. Adding non-specific proteins of similar molecular weight can have a “carrier” effect and substantially increase transfer efficiency.

■ For proteins <100 kDa, try blotting in standard Tris-glycine buffer with 20% methanol and no SDS. Addition of SDS to the transfer buffer can greatly reduce binding of transferred proteins to the membrane (for both PVDF and nitrocellulose).

■ Soak the gel in transfer buffer for 10-20 minutes before setting up the transfer. Soaking equilibrates the gel and removes SDS so that it will not be carried over into the transfer tank.

■ To maximize retention of transferred proteins on the membrane, allow the membrane to air-dry completely after transfer (approximately 1-2 hours).

■ Do not over-block. Long blocking incubations, particularly with nonfat dry milk at 2% or higher, can cause loss of target protein from the membrane (J. Immunol. Meth. 122:129-135, 1989).

■ To enhance signal, try extended primary antibody incubation at room temperature or overnight incubation at 4°C. Avoid extended incubations in secondary antibody.

VII. Imaging of Coomassie-Stained Protein Gels

IRDye Blue Protein Stain is a convenient, safe alternative for gel staining. Unlike traditional Coomassie® Blue stains, which require methanol and acetic acid for staining and destaining, IRDye Blue Protein Stain is water-based and requires no hazardous solvents. This stain offers excellent detection sensitivity in the 700 nm channel of the Odyssey® and Aerius® Imaging Systems (≤ 5 ng of BSA can be detected). IRDye Blue Protein Stain is Coomassie based and is provided as a ready-to-use 1X solution. Prewashing and destaining steps are performed in water.

Protocol:

1. Wash gels with deionized water for 15 minutes.

2. Submerge gel in IRDye Blue Stain for 1 hour.

3. Destain with water for 30 minutes or overnight if needed.

4. After removing the gel, carefully clean the glass scanning surface to remove residual stain.

5. Scan on Odyssey using protein gel preset in Odyssey software and scan in 700 channel only. (Alternatively, the focus offset needs to be set to one-half the thickness of the gel.)

LI-COR Biosciences


VIII. Troubleshooting Guide

Problem Possible Cause Solution / Prevention

High background, uni-formly distributed.

BSA used for blocking. Blocking solutions containing BSA may cause high membrane background. Try adding SDS to reduce background, or switch to a different blocker.

Not using optimal blocking reagent.

Compare different blocking buffers to find the most effective for your system; try blocking longer.

Background on nitrocellulose. Add Tween-20 to the diluted antibodies to reduce background. Try adding SDS to diluted secondary antibody.

Background on PVDF. Reduce Tween-20 in diluted antibodies to 0.1%. Add 0.01-0.02% SDS to diluted secondary antibody.

Antibody concentrations too high.

Optimize primary and secondary anti-body dilutions.

Insufficient washing. Increase number of washes and buffer volume.

Make sure that 0.1% Tween-20 is present in buffer and increase if needed. Note that excess Tween-20 (0.5-1%) may decrease signal.

Cross-reactivity of antibody with contaminants in blocking buffer.

Use Odyssey Blocking Buffer instead of milk. Milk is usually contaminated with IgG and will cross-react with anti-goat secondary antibodies.

Inadequate antibody volume used.

Increase antibody volume so entire membrane surface is sufficiently cov-ered with liquid at all times (use heat-seal bags if volume is limiting). Do not allow any area of membrane to dry out.

Use agitation for all antibody incubations.

Membrane contamination. Always handle membranes carefully and with forceps. Do not allow mem-brane to dry. Use clean dishes, bags, or trays for incubations.

Odyssey® Western Blot Analysis


Uneven blotchy or speck-led background.

Blocking multiple membranes together in small volume.

If multiple membranes are being blocked in the same dish, ensure that blocker volume is adequate for all membranes to move freely and make contact with liquid.

Membrane not fully wetted or allowed to partially dry.

Keep membrane completely wet at all times. This is particularly crucial if blot will be stripped and re-used.

If using PVDF, remember to first pre-wet in 100% methanol.

Contaminated forceps or dishes. Always carefully clean forceps after they are dipped into an antibody solu-tion, particularly dye-labeled second-ary antibody. Dirty forceps can deposit dye on the membrane that will not wash away.

Use clean dishes, bags or trays for incu-bations.

Dirty scanning surface or sili-cone mat.

Clean scanning surface and mat care-fully before each use. Dust, lint, and residue will cause speckles.

Incompatible marker or pen used to mark membrane.

Use only pencil or Odyssey pen to mark membranes.

Weak or no signal. Not using optimal blocking reagent.

Primary antibody may perform substan-tially better with a different blocker.

Insufficient antibody used. Primary antibody may be of low affin-ity. Increase amount of antibody or try a different source.

Extend primary antibody incubation time (try 4 - 8 hr at room temperature, or overnight at 4°C).

Increase amount of primary or second-ary antibody, optimizing for best per-formance.

Try substituting a different dye-labeled secondary antibody.

Primary or secondary antibody may have lost reactivity due to age or stor-age conditions.


LI-COR Biosciences


Weak or no signal(continued)

Too much detergent present; sig-nal being washed away.

Decrease Tween-20 and/or SDS in diluted antibodies. Recommended SDS concentration is 0.01 - 0.02%, but some antibodies may require an even lower concentration.

Insufficient antigen loaded. Load more protein on the gel. Try using the narrowest possible well size to con-centrate antigen.

Protein did not transfer well. Check transfer buffer choice and blot-ting procedure.

Use pre-stained molecular weight marker to monitor transfer, and stain gel after transfer to make sure proteins are not retained in gel.

Protein lost from membrane dur-ing detection.

Extended blocking times or high con-centrations of detergent in diluted anti-bodies may cause loss of antigen from the blotted membrane.

Proteins not retained on mem-brane during transfer.

Allow membrane to air dry completely (1 - 2 hr) after transfer. This helps make the binding irreversible.

Addition of 20% methanol to transfer buffer may improve antigen binding. Note: methanol decreases pore size of gel and can hamper transfer of large proteins.

SDS in transfer buffer may interfere with binding of transferred proteins, especially for low molecular weight proteins. Try reducing or eliminating SDS. Note: presence of up to 0.05% SDS does improve transfer efficiency of some proteins.

Small proteins may pass through mem-brane during transfer (“blow-through”). Use membrane with smaller pore size or reduce transfer time.


Odyssey® Western Blot Analysis



Nonspecific or unexpected bands.

Antibody concentrations too high.

Reduce the amount of antibody used.

Reduce antibody incubation times.

Increase Tween-20 in diluted antibodies.

Add or increase SDS in diluted second-ary antibodies.

Not using optimal blocking reagent.

Choice of blocker may affect background bands. Try a different blocker.

Cross-reactivity between anti-bodies in a two-color experi-ment.

Double-check the sources and specific-ities of the primary and secondary anti-bodies used (see III. Guidelines for Two-Color Detection).

Use only highly cross-adsorbed sec-ondary antibodies.

There is always potential for cross-reac-tivity in two-color experiments. Use less secondary antibody to minimize this.

Always test the two colors on separate blots first so you know what bands to expect and where.

Avoid using mouse and rat antibodies together, if possible. Because the spe-cies are so closely related, anti-mouse will react with rat IgG to some extent, and anti-rat with mouse IgG. Sheep and goat antibodies may exhibit the same behavior.

Bleedthrough of signal from one channel into other channel.

Check the fluorescent dye used. Fluo-rophores such as Alexa Fluor® 750 may appear in both channels and are not recommended for use with Odyssey.

If signal in one channel is very strong (near or at saturation) it may generate a small amount of bleedthrough signal in the other channel. Minimize this by using a lower scan intensity setting in the problem channel.

Reduce signal in further experiments by reducing the amount of protein loaded or antibody used.

4308

Progressive Avenue • P.O. Box 4000 • Lincoln, Nebraska 68504 USATechnical Support: 800-645-4260

North America: 800-645-4267International: 402-467-0700 • 402-467-0819

LI-COR GmbH (Germany, Austria, Switzerland, Czech Republic, Hungary, Slovakia): +49 (0) 6172 17 17 771LI-COR UK Ltd.: +44 (0) 1223 422104

www.licor.com

LI-COR is an ISO 9001 registered company. © 2007 LI-COR Inc. LI-COR, Odyssey, Newblot and IRDye are trademarks or registered trademarks of LI-COR, inc. Alexa Fluor is a registered trademark of Invitrogen Corporation. Coomassie is a trademark of Imperial Chemical Industries PLC. Tween is a registered trademark of ICI Americas, Inc. Cy is a registered trademark of Amersham Pharmacia Biotech. The Odyssey Infrared Imager and the Odyssey system are covered by U.S. patents, foreign equivalents, and patents pending.

®

Odyssey


®

®

In-Gel Western Detection

Doc# 988-08329

Published January, 2006. The most recent version of this protocol is posted at http://biosupport.licor.com/support

Odyssey

®



Contents

Page


II. Description........................................................................................................1

III. Guidelines for Two-Color Western Detection ....................................................2

IV. Electrophoresis .................................................................................................3

V. In-Gel Western Detection Protocol ...................................................................4

VI. Optimization......................................................................................................5

VII. Stripping and Reprobing Gels ..........................................................................7

VIII. Troubleshooting Guide......................................................................................8


Odyssey

®

Reagents

• Infrared dye labeled secondary antibodies (LI-COR)*.

Additional Reagents

• SDS-PAGE gel for electrophoresis.• 50% isopropanol + 5% acetic acid (made with ultrapure water).• Blocking buffer (5% BSA).• Primary antibody.• Tween

®

-20• PBS buffer• Ultrapure water

*IRDye™ 800CW-labeled secondary antibodies are also available from Rockland Immunochemicals, Inc. Alexa Fluor

®

680-labeled secondary antibodies are available from Invitrogen Corporation.

II. Description

Western blot detection of proteins requires separation of protein mixtures by electrophoresis, followed by transfer of the separated proteins to nitrocellulose or PVDF membranes for detection. The Odyssey

®

System allows you to detect target proteins while still embedded in the gel, without transfer to a membrane. This powerful technique saves time, reduces cost, and eliminates the variables introduced by the transfer step or subsequent blocking of the membrane. In-Gel Western detection can be performed with standard Odyssey reagents – no special kit is required.

After electrophoresis, the gel is fixed briefly in a solution of isopropanol and acetic acid. Following a wash step to remove the alcohol, the gel is incubated in diluted antibodies and washed in a manner similar to an ordinary Western blot. After washing, the wet gel is ready to scan on Odyssey. There is no substrate to apply, no plastic wrap, and no film exposures. Two-color Western detection of two different protein targets can be performed within the gel.

LI-COR Biosciences


In-gel detection can enable you to get your results faster. It can also ensure that results are more accurate, because there are no inconsistencies caused by transfer. If your target proteins don’t transfer well (for exam-ple, large proteins that won’t come out of the gel or small proteins that go through the membrane during transfer), in-gel detection bypasses these problems. Gels can be stripped and re-detected if desired.

Odyssey in-gel Westerns also offer unparalleled in-gel detection sensitivity in the low-picogram range. This technique provides a very useful tool for protein detection and research. However, it is important to note that in-gel detection may not be quantitative.

III. Guidelines for Two-Color Western Detection

It is absolutely critical that primary and secondary antibodies be carefully selected for two-color detection or cross-reactivity will result. The following guidelines should be used when selecting primary and second-ary antibodies for two color detection:

a.

All secondary antibodies must be highly cross-adsorbed

to eliminate cross-reactivity.

b.

The two primary antibodies used must be derived from

different

host species

so they can be discrimi-nated by secondary antibodies of different specificities (for example, primaries from rabbit and mouse will be discriminated by anti-rabbit and anti-mouse secondary antibodies).

c.

The two secondary antibodies used must be derived from the

same

host species

so they will not react against one another. The secondary antibodies should not recognize immunoglobulins from other species that may be present in the sample.

d. One secondary antibody should be labeled with IRDye™ 800, and the other with IRDye™ 680 (Alexa Fluor

®

680, Cy

®

5.5, etc.).

e. Always perform preliminary blots with each antibody alone to determine the expected banding pattern for each, before combining them in a two-color experiment. Slight cross-reactivity may occur, particularly if the antigen is very abundant, and can complicate interpretation of your blot. If cross-reactivity is a prob-lem, load less protein or reduce the amount of antibody.

f. For best results, avoid using primary antibodies from mouse and rat together for a two-color experiment. Because the species are so closely related, it is not possible to completely adsorb away cross-reactivity. Substantial cross-reactivity between bands may occur. If using mouse and rat together, it is crucial to run single-color blots first with each individual antibody to be certain of expected band sizes.

Protocol Modifications for Two-Color Detection

For two-color detection, follow the standard protocol given below with the following modifications:

1. Use two labeled secondary antibodies that are labeled with different dyes.

2. Make sure that antibody specificities and hosts are appropriate and will not cross-react.

3. Combine the two primary antibodies in the diluted antibody solution in step 5, and incubate simultaneously with gel.

4. Combine the two dye-labeled secondary antibodies in the diluted antibody solution in step 8. Incubate simultaneously with gel.

Odyssey

®



IV. Electrophoresis

V. In-Gel Western Detection Protocol

1. Separate the proteins of interest by electrophoresis.

Notes:

• Gel type will affect the success and sensitivity of in-gel Western detection. We obtain the best results with NOVEX

®

Tris-glycine pre-cast gels. Other gel types may require optimization.

• Gel thickness and acrylamide percentage affect the ability of antibody molecules to penetrate the gel. We generally recommend that gel percentage be 12% or less, with a gel thickness of 1 - 1.5 mm.

• The performance of different pre-cast gels may vary widely. We recommend NOVEX

®

Tris-glycine gels for maximum performance, but other gel types may be used.

2. Following electrophoresis, separate the two plates. The stacking gel will exhibit high background when gel is scanned. If desired, cut away the stacking gel from the top of the gel using a scalpel or razor blade. Notch one corner of the gel for orientation.

3. Incubate the gel in 50% isopropanol + 5% acetic acid (prepared with ultrapure water) for 15 minutes. Use enough solution that the gel is completely covered and can move freely. Shake gently.

4. Remove isopropanol/acetic acid and wash the gel in ultrapure water for 15 minutes with gentle shaking. Use enough water that the gel is completely submerged and can move freely. The gel may curl and/or float to the surface. Gently flatten the gel or turn it over, making sure that it is completely covered. Residual alcohol on the gel surface can cause diffuse bands.

■

Tip:

If desired, you may stop here and store the gel overnight in water at 4°C.

5. No blocking step is required before antibody incubations. Dilute primary antibody to desired concentration in Odyssey Blocking Buffer, PBS, or 5% BSA (we find that BSA works best). Include 0.1% Tween-20 in the diluted antibody solution. Since in-gel detection is not as sensitive as a standard Western blot, you may wish to use more primary antibody than usual. Make sure the gel is completely covered by antibody solution. Incubate gel in primary antibody for 1 hour with gentle shaking.

6. Primary antibody incubation can be extended to several hours, or carried out overnight at 4 °C. Extended incubation will increase signal.

7. Wash the gel 3 times for 10 minutes in PBS + 0.1% Tween-20 with gentle shaking, using a generous amount of wash buffer.

8. Dilute secondary antibody at 1:1000 – 1:5000 in the appropriate blocker with 0.1% Tween-20. Incubate gel in secondary antibody for 1 hour with gentle shaking, and protect from light. Use enough antibody solution to completely cover gel.

Important: Handle the gel gently. Squeezing or pressing can cause splotches or fingerprints to appear in the image.

LI-COR Biosciences


VI. Optimization

The in-gel detection protocol may require optimization for your target proteins or gel type. However, you should expect the detection sensitivity of in-gel scans to be somewhat lower than a standard Western blot. Transfer to a membrane concentrates the target protein, whereas in gels, protein is dispersed through the thick-ness of the gel.

Use the following guidelines for optimization:

■

Optimization of primary and secondary antibody dilutions, as well as amounts of Tween-20 in diluted anti-bodies, may be needed to achieve maximum signal and minimum background.

■

Try different buffers for dilution of your antibodies, including PBST alone, Odyssey Blocking Buffer, milk, BSA, Pierce SuperBlock

®

, etc. Different blockers can make a big difference. BSA often works best for us (it’s not generally recommended for blocking of membranes for Odyssey, but it works well for in-gel Westerns.)

VII. Stripping and Reprobing

In-gel Westerns may be stripped and re-probed. The recommended stripping buffer is 25 mM glycine-HCl pH 2.0 + 1 – 2% SDS. Strip the gel for 30 - 60 minutes by shaking it in a generous amount of stripping buffer at room temperature. Replace the stripping buffer with fresh buffer every 15 - 30 minutes. Wash the gel extensively in PBS + 0.1% Tween-20 after stripping. To monitor stripping, scan the gel quickly at low resolution (337 µm). The time required to completely strip the gel will depend on the intensity of your orig-inal signal, and how strongly your primary antibody binds its antigen.

9. Wash the gel 3 times for 10 minutes in PBS + 0.1% Tween-20 with gentle shaking, using a generous amount of wash buffer.

10. Wash the gel for 5 minutes in PBS.

11. Lay the wet gel on the scanning surface of the Odyssey instrument. The gel can be scanned uncovered, or covered with plastic wrap to prevent drying. Set the focus offset to 1/2 the gel thickness (for a 1 mm gel, set the focus offset to 0.5 mm). If the stacking gel was not removed from the top of the gel, it can be cropped from the image when creating a new analysis in Odyssey software.

12. If image background is high, the background may be reduced by soaking the gel overnight in PBS and re-scanning. Store the gel at 4 °C and protect from light. Gels can be kept at 4°C for several days, if desired.

Odyssey

®



VII. Troubleshooting Guide


High background in gel. Stacking gel is still present. Cut the stacking gel away after electrophoresis.

Too much antibody. Reduce concentration of secondary antibody.

Uneven gel background may result from insufficient solution volumes for incubations.

Use enough solution at each step (fixation, washes and antibody incubations) to allow the gel to move freely.

Pressing or squeezing gel during fixation and staining can cause splotchy background.

Handle gel gently, with a light touch, and by the edges whenever possible.

Gel was not thoroughly washed. Use plenty of wash buffers to allow gel to move freely. Do not allow the gel to stick to bottom of container.

Extend wash times or increase number of washes. Background may decrease if the gel is allowed to soak in PBS over night at room temp (protect from light).

Weak or no signal. Not enough antibody. Increase amount of primary and/or secondary antibody. Extend primary antibody incubation to overnight at 4 °C to increase signal.

Remember that in-gel detection is not as sensitive as blot detection; adjust sample loading and antibody concentrations accordingly.

Antibody dilution buffer is not optimal for your primary antibody.

Try a different dilution buffer; this can significantly affect performance of some primary antibodies.

Suggested buffers include 3-5% BSA, Odyssey Blocking Buffer and PBS or TBS (all with 0.1% Tween-20). Other blockers (milk, casein, commercial blockers) and Tween-20 concentrations can also be tested.

LI-COR Biosciences


Weak or no signal(Continued).

Gel type is not optimal. We recommend NOVEX pre-cast gels for in-gel detection. Other gel sources and homemade gels can be used, but may show reduced sensitivity and require further optimization.

Antibody did not penetrate gel sufficiently or evenly.

Acrylamide percentage was too high. Try a lower percentage or a gradient gel.

Increase volume for antibody incubations so that gel is completely bathed in antibody.

Make sure gel is adequately fixed. Some monoclonal antibodies may be sensitive to residual acid in the gel; in this situation, eliminate acetic acid from the fix or extend the water wash step.

Gel was left in isopropanol/acetic acid too long.

This may cause protein to be lost from the gel. Fix for 15 minutes only.

Fuzzy or irregularly shaped bands.

Gel type is not optimal. We recommend NOVEX pre-cast gels for in-gel detection. Other gel sources and homemade gels can be used, but may show reduced sensitivity and require further optimization.

Gel is overloaded. Try loading less protein; bands can appear "blobby" if the amount of target protein in the band is too high.

Inadequate fixation of gel. If problem persists when gel is fixed according to the protocol, try adjusting isopropanol or acetic acid concentrations. Fixing in isopropanol alone (no acetic acid) can cause irregularly shaped bands.


Odyssey

®



Nonspecific or unexpected bands.

Antibody concentration too high. Reduce amount of antibody used or reduce incubation times.

Cross-reactivity between antibodies in a two-color experiment.

Antibodies must be chosen carefully. Read

III. Guidelines for Two-Color Western Detection

.

Antibody dilution buffer is not optimal for your primary antibody.

Try a different dilution buffer; this can significantly affect performance of some primary antibodies.

Suggested buffers include 3-5% BSA, Odyssey Blocking Buffer, and PBS or TBS (all with 0.1% Tween-20). Other blockers (milk, casein, commercial blockers) and Tween-20 concentrations can also be tested.

Bleed through between 700 nm and 800 nm channels.

If signal is extremely strong (saturated), it may appear faintly in the other channel. Re-scan gel at a lower intensity or repeat using less antibody.


4308



LI-COR GmbH (Germany, Austria, Switzerland, Czech Republic, Hugary, Slovakia): +49 (0) 6172 17 17 771LI-COR UK Ltd.: +44 (0) 1223 422104

www.licor.com

LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, inc. Alexa Fluor is a registered trademark of Invitrogen Corporation. Tween is a registered trademark of ICI Americas, Inc. Cy is a registered trademark of Amersham Pharmacia Biotech. NOVEX is a registered trademark of Novel Experimental Technology Corporation. SuperBlock is a reg-istered trademark of Pierce Chemical Company. The Odyssey Infrared Imager and the Odyssey system are covered by U.S. patent (6,495,812), foreign equivalents, and patents pending.

®

Odyssey


®

®

Northern Blot Analysis

Using Biotin PCR Labeled Probes

Doc# 988-09394

Published January, 2006. Last updated October, 2007. The most recent version of this protocol is posted athttp://biosupport.licor.com/support

Odyssey

®



Contents

Page


II. Northern Blotting Methods................................................................................2

III. Biotin Probe Labeling Using PCR Amplification ...............................................3

IV. Northern Blot Hybridization ..............................................................................4

V. Biotin Detection for Northern Blots ...................................................................5

VI. Troubleshooting Guide......................................................................................8


Northern Blotting and Hybridization

• Biodyne

®

B Nylon Membranes (Pall, Cat. #60200).• LI-COR recommends the NorthernMax

®

Kit (Ambion, Cat. #1940) for formaldehyde gels.• RNA Loading Buffer (Sigma, Cat. #R-4268).• ULTRAhyb™–Oligo (Ambion, Cat. #8663). This solution has been found to have best performance with

infrared probes. Other hybridization solutions may cause high background.• Sterile DEPC treated water.

Biotin Probe Labeling and Detection

• PCR Amplification Reagents. • Biotin-16-dUTP (Roche, Cat. # 1 093 070). *• Odyssey

®

Blocking Buffer (LI-COR, Cat. #927-40000).• Streptavidin IRDye

®

800CW conjugate (LI-COR, Cat. #926-32230) or Streptavidin IRDye

®

680 conju-gate (LI-COR, Cat. #926-32231)

• QIAQuick

®

PCR Purification Kit, 50 reactions (Qiagen, Cat. #28104) • 20% SDS• 1X PBST (0.1% Tween

®

-20)• 1X PBS

* LI-COR Biosciences recommends that all restrictions placed on product labels and product inserts for biotin-16-dUTP be followed. Applications other than those recommended on the product insert may require a license under certain patents owned by third parties. LI-COR Biosciences does not grant any additional license to make, use or sell this product.

LI-COR Biosciences


II. Northern Blotting Methods

This is a modified version of the NorthernMax

®

protocol for Northern blotting. LI-COR’s system has been optimized using the NorthernMax Kit. Other transfer systems should work provided:

a. Biodyne B Nylon Membranes are used;

b. The loading buffer contains only small amounts of bromophenol blue.

Biodyne B Nylon Membranes work well because they have been tested for reduced infrared background using both IRDye and Biotin labeling methods. Bromophenol blue is detected by Odyssey and can cause high background. Small amounts of the dye can be removed during prehybridization.

IMPORTANT: All equipment must be RNase free and dilutions should be made with RNase-free water to prevent RNA degradation.

Gel Electrophoresis

Transfer

Cross-link

1. Separate RNA on a denaturing formaldehyde MOPS gel, as described in the Ambion NorthernMax protocol.

■

Tip:

Ethidium bromide in large amounts increases background. If ethidium bromide is required, use Sigma loading buffer (Cat. #R-4268) since it does not cause excess background but contains the ethidium bromide necessary for sample visualization. This loading buffer works well for RNA markers.

2. Transfer for 2 hours or more using the NorthernMax transfer buffer.

3. Cross-link RNA onto nylon using a UV crosslinker, or bake at 80°C for 30 minutes.

Important: Do not touch the membrane; always handle by the corners and only with clean forceps. Fingerprints, even from a glove, will clearly show on the scanned image of the membrane.

Odyssey

®



III. Biotin Probe Labeling Using PCR Amplification

This modified biotin labeling protocol is designed to fit directly into any Northern protocol; however, sys-tem optimization may be necessary.

PCR Probe Amplification and Biotin-16-dUTP Incorporation

1. In the PCR reaction, replace the dTTP with 60% unmodified dTTP and 40% biotin-16-dUTP as illustrated below in an example PCR reaction using M13 primers:

Component

DNA 10 ngM13F (50 pM) 0.5 µlM13R (50 pM) 0.5 µl10X Buffer 2.5 µlMgCl

2

(25 mM) 5.0 µldATP (10 mM) 0.625 µldCTP (10 mM) 0.625 µldGTP (10 mM) 0.625 µldTTP (10 mM) 0.375 µl (60%)

←←←←

Biotin-16-dUTP (1 mM) 2.5 µl (40%)

←←←←

Taq

®

Polymerase (5µ/µl) 0.25 µlH

2

O __ µl

TOTAL VOLUME 25.0 µl

2. Amplify the probe using the standard PCR protocol for your specific product. An example program for M13 primers is given below.

Important: For Northern detection to be a success, it is essential to optimize probe amplification and Biotin-16-dUTP incorporation. Each users’ system will be different.

Program:

Cycles Temperature (°C) Time

1 94 6 minutes

30 9545 72

1 minute2 minutes3 minutes

1 72 10 minutes

1 4 hold

LI-COR Biosciences


Visualization on an agarose gel will confirm adequate probe amplification.

If no product can be visual-ized, do NOT proceed with probe purification or Northern blot hybridization.

PCR reaction must be opti-mized before continuing. If the visualized PCR is not a clean fragment or multiple fragments are present, gel extraction and purification of the appropriate size fraction is advised.

Probe Purification

We recommend using QiaQuick

®

PCR Purification Kit (Qiagen, Cat # 28106).

IV. Northern Blot Hybridization

Pre-hybridization

3. Before proceeding with probe purification, run 5 µl of the PCR amplified product on an 0.8% agarose gel and visualize using a UV transilluminator.

4. Add 125 µl of Buffer PB to sample tube. Mix well and add to column. Centrifuge at 12,000 xg for 1 minute. Discard flow through.

5. Add 750 µl of Buffer PE to column. Make sure ethanol is added to the PE buffer before it is used. Centrifuge as in step 4 and discard flow through. Centrifuge again to remove excess PE buffer. Place column into a clean RNase-free centrifuge tube.

6. Add 20 µl of nuclease-free water warmed to 65°C directly to the center of the column to elute. Let stand at room temperature 5 minutes. Centrifuge as above. Repeat elution twice (total of three times).

1. Place blot in hybridization bottle or bag.

2. Pre-hybridize Northern blot for a minimum of 1 hour at 42°C in Ambion ULTRAhyb™–Oligo Buffer, 5 ml per 10 x 10 cm blot.

■

Tip:

Hybridization stringency can be increased or decreased by the hybridization temperature; how-ever, we recommend 42°C as a starting point.

Important: Do not use the ULTRAhyb buffer provided in the NorthernMax Kit. UlTRAhyb buffer is designed for RNA probes. Use only ULTRAhyb–Oligo hybridization solution (Ambion, Cat. #8663) for Odyssey Northern Blots. ULTRAhyb–Oligo has been found to have best performance with infrared probes. Other hybridization solutions may cause high background.

Odyssey

®



Denature Probe

Hybridization

Stringency Washes

V. Biotin Detection for Northern Blots

Blocking

3. The first time a probe is used, hybridize with the entire PCR product. Optimization can be done to reduce the amount of probe per hybridization. No less than 500 ng should be used initially.

4. Denature probe for 5-10 minutes at 95°C and place immediately on ice.

5. Pour pre-hybridization solution off of blot.

6. Add freshly denatured probe directly into fresh ULTRAhyb™–Oligo hybridization solution. Do not use more than 3-5 ml per 10 x 10 cm blot of hybridization solution for each probe.

■

Tip:

The correct probe concentration is essential in obtaining optimal results. If larger volumes are used, the amount of probe must be adjusted accordingly. This step usually needs to be optimized for each user’s system.

7. Add hybridization solution containing probe to the bottle or bag containing blot.

8. Hybridize overnight at 42°C. Time can vary for each sample. Shorter times are possible.

9. Remove hybridization solution and wash at room temperature in NorthernMax Low-Stringency Wash Solution for 5 minutes. Repeat.

10. Wash 15 minutes at 50°C in NorthernMax High Stringency Wash Solution. Repeat.

■

Tip:

Start with 50°C, then increase temperature in small increments if necessary.

1. Add 5 ml of 20% SDS to 95 ml Odyssey Blocking Buffer for a final concentration of 1% SDS.

Important: Always remove the prehybridization solution and replace with fresh hybridization solution. Bromophenol blue from the loading buffer washes off into the pre-hybridization solution and may cause excess background if the pre-hybridization solution is not replaced before overnight hybridization.

Important: Do not to touch the pipette tip or probe directly onto the blot.

Important: Failure to add SDS will result in very high background on blots.

LI-COR Biosciences


Streptavidin Incubation

Wash

Scan Blot On Odyssey

Chapter 3 of the Odyssey Operator’s Manual describes how to place the blot on the Odyssey scanning sur-face. Chapter 2 of the Odyssey User Guide describes how to start scans and set the scanning parameters.

2. In a container, cover blot with Odyssey Blocking Buffer plus SDS and gently shake at room temperature for a minimum of 30 minutes. For more sensitive detection, blocking for a longer time may reduce background.

3. Dilute Streptavidin-IRDye 680 conjugate or Streptavidin- IRDye 800CW conjugate with Odyssey Blocking Buffer plus 1% SDS to a concentration of 1:10,000.

4. Remove old blocking buffer and lightly cover the blot with the 1:10,000 streptavidin-IRDye 680 or Streptavidin-IRDye 800CW buffer, approximately 5 ml/10 cm

2

. Incubate 30 minutes at room temperature while gently shaking.

5. Wash the blot 3 times in 1X PBST (0.1% Tween-20) for 5 minutes each, shaking at room temperature. Follow with a rinse in 1X PBS for 5 minutes at room temperature. Wash steps must be performed in darkness. Use a black dish or cover container with aluminum foil.

6. Scan blot on Odyssey. Start with the

Intensity

parameter set to 5 for Northern blots. If necessary, scan again and adjust intensity.

Important: This reaction is light-sensitive; it is essential to incubate in darkness.

Odyssey

®



VI. Troubleshooting Guide


Low sensitivity (faint bands or no bands).

Insufficient hybridization time. For most applications, hybridize overnight.

Incomplete transfer. Following RNA transfer to membrane, view the gel with UV transilluminator to see if any RNA has remained in the gel.

RNA degraded. Use DEPC or Nuclease free water for all dilutions.

Keep all plastics and glassware RNase free.

Target DNA not effectively fixed on membrane.

Check UV lamp or oven temperature.

Poorly labeled probe. Visualize PCR incorporated probe on an agarose gel to verify adequate amplification and incorporation of biotin-16-dUTP.

Low probe concentration. Probe concentrations vary. Quantify PCR product to verify probe concentration.

Make sure you added the ethanol to the wash buffer in the cleanup kit.

Increase the amount of probe used in the hybridization reaction.

Low hybridization efficiency. Increase hybridization time or probe concentration.

Low target concentration. Increase amount of target RNA.

Verify that RNA was not degraded by visualizing on gel before transfer.

Too high stringency. Decrease time or temperature of stringency washes.

RNA on membrane inaccessible to probe.

Place membrane in tube or bag with RNA side exposed to the hybridization solution.

LI-COR Biosciences


Low sensitivity (continued).

Intensity set too low when the scan is started.

Increase the intensity settings on the Scanner Console window by increments of 0.5 in one or both channels. Re-scan membrane.

If the intensity was only slightly low during scanning, images can be visually modified using the "alter intensity" dialog box. Sensitivity, brightness and contrast can be adjusted.

Not enough streptavidin. Increase the amount of streptavidin used in the detection steps.

Uneven, blotchy, speckled, or high background.

Membrane contamination. Always handle the membranes by the edges and only with forceps. Fingerprints, even from a glove, cause increased background.

Insufficient pre-hybridization of nylon.

Use adequate hybridization buffer to cover membranes and possibly extend the prehybridization time.

Ensure that hybridization solution is pre-warmed and completely in solution before using.

Make sure to use ULTRAhyb™–Oligo (Ambion, Cat. #8663). It has been optimized for the Odyssey system.

Contaminated forceps or dishes. Always clean forceps after they are used with hybridization solutions containing labeled probe. Dirty forceps may deposit dye on membrane that will not wash away.

Use clean dishes, bags, or bottles for incubations.

Hybridizing or washing multiple membranes together in a small volume.

When hybridizing multiple membranes, make sure they do not overlap and there is enough hybridization solution to cover the membranes.

When washing membranes together, provide enough wash solution to allow the membranes to move freely in the dish.


Odyssey

®



Uneven, blotchy, speckled, or high background (continued).

Too low of stringency or not long enough wash time.

Increase time of stringency wash to remove background signal.

Increase temperature of stringency wash.

Membrane not fully wetted or has become partially dry.

Keep membrane completely wet after hybridizing. This is particularly crucial if blot will be stripped and re-used.

Do not allow the membrane to dry between pre-hybridization and hybridization.

Probe added onto membrane. Add the labeled probe to the hybridization solution. Avoid touching the membrane with the pipette tip containing the labeled probe.

Too much labeled probe. Decrease the amount of labeled probe added to the hybridization. This reduces background while retaining sensitivity.

Incorrect loading buffer used. Use RNA Loading Buffer (Sigma, Cat. #R-4268). Bromophenol blue and other dyes cause background fluorescence.

Inadequate PCR amplification. Visualize the PCR incorporated probe on an agarose gel. If the fragment is not a sharp band or there are multiple fragments present, gel extract the appropriate fragment, purify, and use that as the probe instead of the entire PCR reaction.

Use sequence or gene specific primers for PCR amplification rather than vector related primers (example: M13). If this is not possible, digest PCR reaction to cleave off the vector sequence and gel purify insert.

PCR amplified probe was not purified.

Purify PCR reaction.

SDS was NOT added to the Odyssey Blocking Buffer.

Add 1% SDS to the Odyssey Blocking Buffer before using in blocking and detection steps of protocol.


LI-COR Biosciences



Inadequate washing following streptavidin conjugation.

Increase PBST wash time following streptavidin conjugation.

Inadequate blocking time before addition of streptavidin.

Increase blocking time before streptavidin conjugation; make sure to use fresh blocking reagent in the streptavidin conjugation step.

Too much streptavidin in conjugation step.

Reduce the amount of streptavidin used in conjugation step.


4308




www.licor.com

LI-COR is an ISO 9001 registered company. © 2006-2007 LI-COR Inc. LI-COR, Odyssey, and IRDye are registered trademarks of LI-COR, inc. Biodyne is a registered trademark of Pall Corporation. Alexa Fluor is a registered trademark of Invitrogen Corporation. NorthernMax and Ultrahyb are trademarks or registered trademarks of Ambion Inc. QIAQuick is a registered trademark of Qiagen. Tween is a registered trademark of ICI Amer-icas, Inc. Taq is a registered trademark of Amersham Pharmacia Biotech Corp. The Odyssey Infrared Imager and the Odyssey system are covered by U.S. patent (6,495,812), foreign equivalents, and patents pending.

®

Odyssey


®

®

Southern Blot Analysis

Using Biotin-Labeled Probes

Doc# 988-09395

Published January, 2006. Last Updated October, 2007. The most recent version of this protocol is posted at http://biosupport.licor.com/support

Odyssey

®



Contents

Page


II. Making Solutions for Southern Blot Analysis ..................................................10

III. Southern Blotting Methods ...............................................................................2

IV. Biotin Probe Labeling Using PCR Amplification ...............................................3

V. Southern Blot Hybridization ..............................................................................4

VI. Biotin Detection for Southern Blots...................................................................6

VII. Troubleshooting Guide......................................................................................7


Southern Blotting and Hybridization

• Biodyne

®

B Nylon Membranes (Pall, Cat. #60200)• DNA hybridization solution (see

II. Making Solutions for Southern Blot Analysis

)• Wash Solutions #1, #2, and #3 (see


)• Sheared and denatured salmon sperm DNA• 100X Denhardt’s solution

Biotin Probe Labeling and Detection

• PCR Amplification Reagents• Biotin-16-dUTP (Roche, Cat. #1 093 070)*• Odyssey

®

Blocking Buffer (LI-COR, Cat. #927-40000)• Streptavidin IRDye

®

800CW, 0.5 mg (LI-COR, Cat. #926-32230**)• QIAquick

®

PCR Purification Kit, 50 reactions (Qiagen, Cat. #28104)• 20% SDS• 1X PBST (0.1% Tween

®

-20)• 1X PBS


** Streptavidin IRDye

®

800CW can also be purchased from Rockland Immunochemicals (Cat. #S000-31).

LI-COR Biosciences



Solutions referred to in this protocol can be made as indicated below. Storage conditions for excess solution are listed.

DNA Hybridization Solution (200 ml; store at 4°C):

10% (w/v) Dextran Sulfate; 5X SSPE; 2% (w/v) SDS

Wash Solution #1 (1L; store at room temperature):

2X SSPE

Wash Solution #2 (1L; store at room temperture):

2X SSPE; 1% (w/v) SDS

Wash Solution #3 (1L; store at room temperature):

0.1X SSPE

III. Southern Blotting Methods

Most Southern blotting systems should work as long as the following guidelines are observed:

a. Biodyne B Nylon Membranes are used; b. The loading buffer contains only small amounts of bromophenol blue.

Biodyne B Nylon Membranes work well because they have been tested for reduced infrared background using both IRDye and Biotin labeling methods. Bromophenol blue is detected by Odyssey and can cause high background. Small amounts of the dye can be removed during prehybridization. Ideally, use a loading buffer that does NOT contain bromophenol blue.

Southern Blotting

Cross-link

1. Prepare membranes for hybridization using Southern or dot/spot/slot blot methods.

■

Tip:

For best performance, use Odyssey reagents for blotting. Hybridization solution and wash solu-tions should be made according to


. Any additional reagents used should be of the highest grade available to reduce background on the membrane. Filter all reagents prior to blotting.

■

Tip:

High concentrations of ethidium bromide in the agarose gel can increase background. If ethid-ium bromide is necessary, soak the gel for 30 minutes to de-stain prior to transfer.

■

Tip:

Use a 6X xylene cyanol loading buffer only (0.1% xylene cyanol/30% glycenol). Dyes such as bromophenol blue fluoresce and cause high background on the membrane. Note that xylene cyanol runs at approximately 700-800 bp. Do not run the dye front past halfway.

2. Cross-link RNA onto nylon using a UV crosslinker, or bake at 80°C for 30 minutes.

Important: Do not touch the membrane; always handle by the corners and only with clean forceps. Fingerprints, even from a glove, will clearly show on the scanned image of the membrane.

Odyssey

®



IV. Biotin Probe Labeling Using PCR Amplification

This modified biotin labeling protocol is designed to fit directly into any Southern protocol; however, sys-tem optimization may be necessary.

PCR Probe Amplification and Biotin-16-dUTP Incorporation

1. In the PCR reaction, replace the dTTP with 60% unmodified dTTP and 40% biotin-16-dUTP as illustrated below in an example PCR reaction using M13 primers:

Component

DNA 10 ngM13F (50 pM) 0.5 µlM13R (50 pM) 0.5 µl10X Buffer 2.5 µlMgCl

2

(25 mM) 5.0 µldATP (10 mM) 0.625 µldCTP (10 mM) 0.625 µldGTP (10 mM) 0.625 µldTTP (10 mM) 0.375 µl (60%)

←←←←

Biotin-16-dUTP (1 mM) 2.5 µl (40%)

←←←←

Taq

®

Polymerase (5µ/µl) 0.25 µlH

2

O __ µl

TOTAL VOLUME 25.0 µl

2. Amplify the probe using the standard PCR protocol for your specific product. An example program for M13 primers is given below.

Important: For Southern detection to be a success, it is essential to optimize probe amplification and Biotin-16-dUTP incorporation. Each user’s system will be different.

Program:

Cycles Temperature (°C) Time

1 94 6 minutes

30 9545 72

1 minute2 minutes3 minutes

1 72 10 minutes

1 4 hold

LI-COR Biosciences


Visualization on an agarose gel will confirm adequate probe amplification.

If no product can be visual-ized, do NOT proceed with purification or Southern blot hybridization.

The PCR reaction must be opti-mized before continuing. If the visualized PCR is not a clean fragment or multiple fragments are present, gel extraction and purification of the appropriate size fraction is advised.

Probe Purification

We recommend using QIAquick

®

PCR Purification Kit (Qiagen, Cat # 28106).

V. Southern Blot Hybridization

Pre-hybridization

3. Before proceeding with purification, it is highly recommended that you run 5 µl of the PCR amplified product on an 0.8% agarose gel and visualize using a UV transilluminator.

4. Add 125 µl of Buffer PB to sample tube. Mix well and add to column. Centrifuge at 12,000 xg for 1 minute. Discard flow through.

5. Add 750 µl of Buffer PE to column. Make sure ethanol is added to the PE buffer before it is used. Centrifuge as in step 4 and discard flow through. Centrifuge again to remove excess PE buffer. Place column into a clean RNase-free centrifuge tube.

6. Add 20 µl of Buffer EB, warmed to 65°C, directly to the center of the column to elute. Let stand at room temperature for 5 minutes. Centrifuge as in step 4. Repeat elution step 2 more times.

1. Add 50 µl of 100X Denhardt’s Solution to 5 ml of DNA hybridization solution.

2. Add 10 µg denatured and sheared salmon sperm DNA per 1 ml DNA hybridization solution containing 1X Denhardt’s Solution.

3. Pre-warm hybridization solution to 65°C.

■

Tip:

Pre-warmed hybridization solution should be completely dissolved. Mix well before using.

4. Place blot in hybridization bottle or bag.

5. Pre-wet the membrane in Wash Solution #1.

6. Pre-hybridize Southern blot for a minimum of 1 hour at 65°C in pre-warmed DNA hybridization solution containing 1X Denhardt’s and salmon sperm DNA (0.1 ml hybridization solution per cm

2

nylon membrane).

■

Tip:

Membranes may be prehybridized longer to decrease background.

■

Tip:

When using larger sized blots, increase the amount of hybridization solution per cm

2

. Use only enough solution to cover the membrane.

Odyssey

®



Denature Probe

Hybridization

The first time a probe is used, hybridize with the entire PCR product. Optimization can be done to reduce the amount of probe per hybridization. No less than 500 ng of PCR product should be used initially.

Stringency Washes

Use clean containers and forceps to avoid cross-contamination and reduce background.

7. Denature probe for 5-10 minutes at 95°C and place immediately on ice.

8. Pour pre-hybridization solution off of blot.

9. Add freshly denatured probe directly into fresh hybridization solution containing both 1X Denhardt’s and salmon sperm DNA. Do not use more than 3-5 ml of hybridization solution per 10 x 10 cm blot.

■

Tip:

The correct probe concentration is essential in obtaining optimal results. If larger volumes are used, the amount of probe must be adjusted accordingly.

This step will need to be optimized for your system.

Start by adding the entire volume of probe.

10. Add hybridization solution containing probe to the bottle or bag containing blot.

11. Hybridize overnight at 65°C.

■

Tip:

Time can vary for each sample. Shorter times are possible; however, there may be a reduction in signal intensity sensitivity. Temperature may be lowered for less stringent conditions and must be optimized for some applications.

12. Carefully remove membrane from the hybridization solution and place membrane in a clean container for washing. Washing may also be performed in the hybridization bottles.

■

Tip:

Multiple membranes can be washed together, provided there is ample volume for each membrane to move freely.

13. Remove hybridization solution and wash twice at room temperature in Wash Solution #1 for 5 minutes.

■

Tip:

Start with 50°C, then increase temperature in small increments if necessary.

14. Wash twice for 15 minutes at 60°C with Wash Solution #2.

■

Tip:

If hybridization was done at a temperature lower than 65°C, the wash temperature should also be lowered to reduce stringency.

15. Wash twice for 15 minutes at 60°C with Wash Solution #3.

Important: Always remove the prehybridization solution and replace with fresh hybridization solution (step 9).

Important: Do not touch the blot with the pipette tip or probe.

LI-COR Biosciences


VI. Biotin Detection for Southern Blots

Blocking

Streptavidin Incubation

Wash

Protect from light during wash steps.

Scan Blot On Odyssey

Chapter 3 of the Odyssey Operator’s Manual describes how to place the blot on the Odyssey scanning sur-face. Chapter 2 of the Odyssey User Guide describes how to start scans and set the scanning parameters.

1. Add 5 ml of 20% SDS to 95 ml Odyssey Blocking Buffer for a final concentration of 1% SDS.

2. In a container, cover blot with Odyssey Blocking Buffer plus SDS and gently shake at room temperature for a minimum of 30 minutes. For more sensitive detection, blocking for a longer time may reduce background.

3. Dilute streptavidin-IRDye 800CW conjugate with Odyssey Blocking Buffer plus 1% SDS to a concentration of 1:10,000.

4. Remove old blocking buffer and cover the blot with a thin layer of diluted streptavidin-IRDye 800CW solution. Use approximately 5 ml of buffer per 10 cm

2

of membrane. Incubate 30 minutes at room temperature while shaking.

5. Wash the blot 3 times in 1X PBST (0.1% Tween-20) with shaking, for 5 minutes each, at room temperature. Follow with a rinse in 1X PBS, with shaking, for 5 minutes at room temperature.

6. Start with intensity setting of 7.

Important: When using this labeling method on Odyssey, it is important that recommended reagents be used for detection.

Important: This step is essential. Failure to add SDS will result in very high background on blots.

Important: This IRDye 800CW conjugate is light-sensitive; protect from light during incubation.

Odyssey

®



VII. Troubleshooting Guide


Low Sensitivity (faint bands or no bands).

Insufficient hybridization time. For most applications, hybridize overnight.

Incomplete transfer. Following DNA transfer to membrane, view the gel with UV transilluminator to see if any DNA has remained in the gel.

Target DNA not effectively fixed on membrane.

Check UV lamp or oven temperature.

Poorly labeled probe. Visualize PCR incorporated probe on an agarose gel to verify adequate amplification and incorporation of biotin-16-dUTP.

Low probe concentration. Probe concentrations vary. Quantify PCR product to verify probe concentration.

Make sure you added the ethanol to the wash buffer in the cleanup kit.

Increase the amount of probe used in the hybridization reaction.

Low hybridization efficiency. Increase hybridization time or probe concentration.

Low target concentration. Increase amount of target DNA used.

Verify that DNA was not degraded on agarose gel before digestion.

Too high stringency. Decrease time or temperature of stringency washes.

Membrane with hybridized target DNA inaccessible to the probe.

Place membrane in tube or bag with DNA side exposed to the hybridization solution.

LI-COR Biosciences


Low Sensitivity (continued).

Intensity set too low on Odyssey when the scan is started.

Increase the intensity settings in the Scanner Console window by increments of 0.5 in one or both channels. Re-scan membrane.

Images with weak signal can be enhanced by selecting Alter Intensity from the View menu and adjusting the sensitivity, brightness or contrast.

Not enough streptavidin. Increase the amount of streptavidin used in the detection steps.

Uneven, blotchy, speckled, or high background.

Membrane contamination. Always handle the membranes by the edges and only with forceps. Fingerprints, even from a glove, cause increased background.

Insufficient pre-hybridization of nylon.

Use adequate hybridization buffer to cover membranes and possibly extend the prehybridization time.

Make sure that hybridization solution is pre-warmed and completely in solution before using.

Contaminated forceps or dishes. Always clean forceps after they are used with hybridization solutions containing labeled probe. Dirty forceps may deposit dye on membrane that will not wash away.

Use clean dishes, bags, or bottles for incubations.

Hybridizing or washing multiple membranes together in a small volume.

When hybridizing multiple membranes, make sure they do not overlap and there is enough hybridization solution to cover the membranes.

When washing membranes together, provide enough wash solution to allow the membranes to move freely in the dish.


Odyssey

®




Too low of stringency or not long enough wash time.

Increase time of stringency wash to remove background signal.

Increase temperature of stringency wash.

Membrane not fully wetted or has become partially dry.

Keep membrane completely wet after hybridizing. This is particularly crucial if blot will be stripped and re-used.

Do not allow the membrane to dry between pre-hybridization and hybridization.

Probe added onto membrane. Add the labeled probe to the hybridization solution. Avoid touching the membrane with the pipette tip containing the labeled probe.

Too much labeled probe. Decrease the amount of labeled probe added to the hybridization. This reduces background while retaining sensitivity.

Incorrect loading buffer used. Use 6X loading buffer (0.1% Xylene Cyanol + 30% glycerol). Bromophenol Blue and other dyes cause background fluorescence.

Inadequate PCR amplification. Visualize the PCR incorporated probe on an agarose gel. If the fragment is not a sharp band or there are multiple fragments present, gel extract the appropriate fragment, purify, and use that as the probe instead of the entire PCR reaction.

Use sequence or gene specific primers for PCR amplification rather than vector related primers (example: M13). If this is not possible, digest PCR reaction to cleave off the vector sequence and gel purify insert.

PCR amplified probe was not purified.

Purify PCR reaction.

SDS was NOT added to the Odyssey Blocking Buffer.

Add 1% SDS to the Odyssey Blocking Buffer before using in blocking and detection steps of protocol.


LI-COR Biosciences



Inadequate washing following streptavidin conjugation.

Increase PBST wash time following streptavidin conjugation.

Inadequate blocking time before addition of streptavidin.

Increase blocking time before streptavidin conjugation making sure to use fresh blocking reagent in the streptavidin conjugation step.

Too much streptavidin in conjugation step.

Reduce the amount of streptavidin used in conjugation step.


4308




www.licor.com

LI-COR is an ISO 9001 registered company. © 2006-2007 LI-COR Inc. LI-COR, Odyssey, and IRDye are registered trademarks of LI-COR, inc. Biodyne is a registered trademark of Pall Corporation. QIAquick is a registered trademark of Qiagen. Tween is a registered trademark of ICI Ameri-cas, Inc. Taq is a registered trademark of Amersham Pharmacia Biotech Corporation. The Odyssey Infrared Imager and the Odyssey system are cov-ered by U.S. patent (6,495,812), foreign equivalents, and patents pending.

®

Odyssey


®

In-Cell Western Assay

For Assessing Response of A431 Cells toStimulation with Epidermal Growth Factor

Revised January, 2006. The most recent version of this protocol is posted at http://biosupport.licor.com/support

®

Doc# 988-08332

Odyssey

®

In-Cell Western Protocol


Contents

Page


II. Seeding, Stimulation, and Detection of A431 Cellular Response to Epidermal Growth Factor ..................................................................................2

III. Experimental Considerations............................................................................5

IV. Experimental Results........................................................................................7


Odyssey

®

Reagents

• IRDye™ 800CW-labeled goat anti-mouse secondary antibodies (LI-COR, Cat.# 926-32210)*• IRDye™ 680-labeled goat anti-rabbit secondary antibodies (LI-COR, Cat.# 926-32221)*

* Other IRDye™ 800CW-labeled secondary antibodies are available from LI-COR and Rockland Immunochemicals, Inc. Alexa Fluor

®


• Odyssey

®

Blocking Buffer (LI-COR, Cat.# 927-40000)

Additional Reagents

• 1X PBS wash buffer.• Tissue culture reagents (serum, DMEM, trypsin, 1X PBS).• 20% Tween

®

-20.• Epidermal Growth Factor (Upstate Group Inc, Cat.# 01-107).• 37% formaldehyde.• 10% Triton

®

X-100.• Nunc™ 96 Microwell™ Plate ( Nunc, Cat.# 167008).• Primary antibodies as described below.

Special Note: Phosphorylated-EGFR and phosphorylated-ERK are purchased from Cell Signaling Technonolgy and Santa Cruz Biotechnology, respectively. Serum starvation of the cells is required to obtain maximal response.

LI-COR Biosciences


II. Seeding, Stimulation and Detection of the A431 Cellular Response to Epidermal Growth Factor

1. Allow A431cell growth in a T75 flask in DMEM and 10% fetal calf serum (FCS; Gibco

®

) using standard tissue culture procedures until cells reach 80%-90% confluency (~1.5x10

7

cells).

2. Remove growth media, wash cells with sterile 1X PBS, and trypsinize cells.

3. Neutralize displaced cells with culture media and pellet by centrifugation.

4. Remove supernatant and resuspend cell pellet in remaining media by manually tapping the collection tube. Avoid vigorous pipetting or vortexing to resuspend cells in order to maintain cell integrity.

5. Dilute cells to 20 ml in complete media and count cells using a hemacytometer.

6. Dilute cells with complete media to concentration of 200,000 cells/ml.

7. Gently mix the cell suspension thoroughly.

8. Under sterile conditions dispense 200 µl of the cell suspension per well in a Nunc™ 96 Microwell™ plate (40,000 cells plated per well).

9. Incubate cells and monitor cell density until cells are consistently confluent in each well. This should take approximately three days.

10. Warm serum free media (D-MEM; Gibco) to 37 °C.

11. Remove complete media from the microwell plate by aspiration.

12. Replace media with 200 µl of pre-warmed serum-free media per well and incubate 4 to 16 hours.

13. In a separate 96-well Microwell™ plate, dispense 100 µl of D-MEM per well.

14. Leave the first and second wells without EGF (resting cells controls). In the remaining wells, add aliquots of a solution of EGF to make serial dilutions ranging 0.2 to 100 ng/ml in the microplate. The experimental layout should look like that shown in Figure 1.

15. Remove starvation media from plate wells by aspiration.

16. Transfer EGF dilutions from the dilution plate into the cell-containing plate.

17. Incubate at 37 °C for 7.5 minutes.

18. Prepare fresh

Fixing Solution

as follows:

1X PBS 45.0 ml37% Formaldehyde 5.0 ml

3.7% Formaldehyde 50.0 ml

Odyssey

®



19. Remove EGF-containing media by aspiration. Immediately fix cells by addition of 150 µl of fresh

Fixing Solution

and incubate at room temperature (RT) for 20 minutes with no shaking.

Add the

Fixing Solution

carefully by pipetting down the side of the wells to avoid detaching the cells.

20. Prepare

Triton Washing Solution

as follows:

1X PBS 495 ml10% Triton X-100 5 ml

1X PBS + 0.1% Triton X-100 500 ml

21. Remove the

Fixing Solution

by aspiration.

22. Wash four times with 200 µl of


for 5 minutes per wash to permeabilize the cells.

Notes:

• Allow each wash to shake on a rotator for 5 minutes at RT.

• Do not allow cells/wells to become dry during washing. Add washes immediately after each other.

23. Remove the


by aspiration.

24. To each well, carefully add 150 µl of LI-COR Odyssey Blocking Buffer (#927-40000) down the side of the wells and incubate for 1.5 hours at RT with moderate shaking on a rotating platform.

Notes:

• No single blocking reagent will be optimal for every antigen-antibody pair. Some primary antibodies may exhibit greatly reduced signal or different nonspecific banding in different blocking solutions. If you have difficulty detecting your target protein, changing the blocking solution may dramatically improve perfor-mance. If the primary antibody has worked well in the past using chemiluminescent detection, try that blocking solution for Aerius detection.

• Odyssey Blocking Buffer often yields higher and more consistent sensitivity and performance than other blockers. Nonfat dry milk or casein dissolved in PBS can also be used for blocking and antibody dilution. Milk-based reagents can interfere with detection when using anti-goat antibodies. They also deteriorate rap-idly at 4°C, so diluted antibodies cannot be kept and re-used for more than a few days. If using casein, a 0.1% solution in 0.2 X PBS buffer is recommended (Hammersten-grade casein is not required).

• Milk-based reagents can interfere with detection when using anti-goat antibodies. They also deteriorate rap-idly at 4°C, so diluted antibodies cannot be kept and re-used for more than a few days.

• Blocking solutions containing BSA can be used, but in some cases they may cause high membrane back-ground. BSA-containing blockers are not generally recommended and should be used only when the pri-mary antibody requires BSA as blocker.

LI-COR Biosciences


25. Dilute the antibodies in LI-COR Odyssey Blocking Buffer to give the concentrations specified below.

Primary antibodies can be added in a variety of combinations. Generally one antibody will be directed against the phosphorylated form of the target protein and the second antibody will be directed against the target protein regardless of phosphorylation status. The following are suggested combinations of primary antibodies depending upon the target to be detected:

a. Phospho-EGFR Tyr1045 (Rabbit; 1:100 dilution; Cell Signaling Technology 2237) Total EGFR (Mouse; 1:500 dilution; Biosource International AHR5062)

b. Phospho-EGFR Tyr1045 (Rabbit; 1:100 dilution; Cell Signaling Technology 2237)Total ERK2 (Mouse; 1:75 dilution; Santa Cruz Biotechnology SC-1647)

c. Phospho-ERK (mouse; 1:100 dilution; Santa Cruz Biotechnology SC-7383)Total ERK1 (Rabbit; 1:200 dilution; Santa Cruz Biotechnology SC-94)

d. Phospho-EGFR Tyr1045 (Rabbit; 1:100 dilution; Cell Signaling Technology 2237)Phospho-ERK (mouse; 1:100 dilution; Santa Cruz Biotechnology SC-7383)

26. Add 50 µl of LI-COR Odyssey Blocking Buffer to one set of wells. These wells will serve as a control for any potential background due to the dye-labeled secondary antibody. See Figure 1 for an example of the desired plate layout.

27. Mix the primary antibody solution well before addition to wells.

28. Remove the blocking buffer by aspiration and add 50 µl of the desired primary antibody combination to the remaining wells. The antibody solution should cover the bottom of each well.

29. Incubate with primary antibody for 2 hours with gentle shaking at RT.

Notes:

• For greatest sensitivity continue incubation overnight at 4 °C with no shaking.

• To avoid the cells drying out, cover the plates if left overnight.

30. Prepare

Tween Washing Solution

as follows:

1X PBS 995 ml20% Tween-20 5 ml

1X PBS with 0.1% Tween-20 1000 ml

31. Wash the plate with


by gently adding buffer down the side of the wells to avoid detaching the cells. Use a generous amount of buffer (200-500 µl). Allow wash to shake gently on a rotator for 5 minutes at RT.

32. Repeat wash 4 more times.

Odyssey

®



III. Experimental Considerations

Proper selection of microplates can significantly affect the results of your analysis, as each plate has its own characteristics including well depth, plate autofluorescence, and well-to-well signal crossover. Use the general considerations for microplate selection provided below.

• In-Cell Western analyses use detection at the well surface with no liquid present. This results in minimal well-to-well signal spread, allowing the use of both clear as well as black-sided plates with clear bottoms.

Do not use plates with white wells, since the autofluorescence from the white surface will create significant noise.

• In Cell Western assays require sterile plates for tissue culture growth. The following plates are recommended by LI-COR Biosciences:

96 well format Nunc™ (Part Number 161093, 165305)96 well format Falcon™ (Part Number 353075, 353948)


33. Calculate the amount of secondary antibody required for the experiment. Dilute the fluorescent-labeled secondary antibodies in Odyssey blocking buffer according to the dilution factors specified below. To lower background, add Tween-20 to the diluted antibody to a final concentration of 0.2%.

a. Goat anti-rabbit IRDye™ 680 (1:200 dilution; LI-COR Cat# 926-32221)b. Goat anti-mouse IRDye™ 800CW (1:800 dilution; LI-COR Cat# 926-32210)

Recommended dilution range is 1:200 to 1:1,200.

34. Mix the antibody solutions thoroughly, add 50 µl of the secondary antibody solution to each well and incubate for 60 minutes with gentle shaking at RT. Protect plate from light during incubation.



(step 30) by gently adding buffer down the side of the wells to avoid detaching the cells. Use a generous amount of buffer (200-500 µl). Allow wash to shake gently on a rotator for 5 minutes at RT.


37. After final wash, remove wash solution completely from wells. Turn the plate upside down and tap or blot gently on paper towels to remove traces of wash buffer. For best results, scan plate immediately; plates may also be stored at 4 °C for up to several weeks (protected from light).

38. Before scanning, clean the bottom plate surface and the Odyssey Imager scanning bed with moist, lint-free paper to avoid obstructions during scanning.

39. Scan the plate simultaneously at 700 nm and 800 nm using the Odyssey Infrared Imaging System. Use medium scan quality, 169 µm resolution, 3.0 mm focus offset, and an intensity setting of 5 for both 700 and 800 nm channels.

Avoid prolonged exposure of the antibody vials to light.

Protect plate from light during washing.

LI-COR Biosciences


• The Odyssey Imager requires that microplates have a maximum 4.0 mm distance from the Odyssey scanning surface to the target detection area of the plate. When using the plates specified above for In-Cell Western assays, the recommended focus offset is 3.0 mm.

• Plates deviating from LI-COR recommendations may require lower or higher focus offsets for optimal resolu-tion and detection. If alternative plates are used, an initial optimization scan will be necessary. Scan a plate containing experimental and control samples at 0.5, 1.0, 2.0, 3.0, and 4.0 mm focus offsets. Use the same intensity settings for each scan. After reviewing the collected scans, use the focus offset with the highest sig-nal-to-noise as your focus offset for experiments using the alternate plates.

• Protect plates from light before imaging to ensure highest sensitivity. When storing plates after imaging, the plates should remain protected from light at room temperature or 4 °C.

• Intensity for both 700 and 800 nm channels should be set to 5 for initial scanning. If your image signal is sat-urated or too high, re-scan using a lower intensity setting (i.e., 2.5). If your image signal is too low, re-scan using a higher intensity setting (i.e., 7.5).

• Scan settings of medium to lowest quality, with 169 µm resolution, provide satisfactory results with minimal scan time. Higher scan quality or resolution may be used, but scan time will increase.

• Establish the specificity of your primary antibody by screening plate-like lysates through Western blotting and detection on the Odyssey instrument. If significant non-specific banding is present, choose alternative pri-mary antibodies to avoid results with non-specific signal detection.

Odyssey

®



IV. Experimental Results

Quantitative and simultaneous measurements of EGFR and phosphorylation

of EGFR in response to EGF stimulation.

Two-color display ofboth 700 and 800 nm

images.

700 nm image(phosphorylated

EGFR).

800 nm image(total EGFR).

0.2 0.4 0.8 1.6 3.2 6.25 12.5 25 50 100

ng/mlBackground Resting

Figure 1. Dose response of A431 cells to Epithelial growth factor (EGF) as measured by specific anti-body detecting phosphorylated EGF receptor (Tyr1045). The image represents a 96-well two color In Cell Western with the 800 and 700 chan-nels detecting total EGF receptor (as normalization) and phosphorylated EGF receptor, respectively. Background wells were incubated with secondary antibody but no primary antibody. The graph repre-sents normalized quantitative data demonstrating the percent phosphorylation of EGF receptor.

LI-COR Biosciences


Quantitative and simultaneous measurements of ERK and phosphorylation

of EGF receptor in response to EGF stimulation.


images.


EGFR).

800 nm image(total ERK).

0.2 0.4 0.8 1.6 3.2 6.25 12.5 25 50 100


Figure 2. Dose response of A431 cells to Epithelial growth factor (EGF) as measured by specific anti-body detecting phosphorylated EGF receptor (Tyr1045). The image represents a 96-well two color In Cell Western with the 800 and 700 chan-nels detecting total ERK (as normalization) and phosphorylated EGF receptor, respectively. Back-ground wells were incubated with secondary anti-body but no primary antibody. The graph represents normalized quantitative data demonstrating the percent phosphorylation of EGF receptor.

Odyssey

®



Quantitative and simultaneous measurements of total ERK and

phosphorylation of ERK in response to EGF stimulation.

l


images.

700 nm image(phosphorylated ERK).

800 nm image(total ERK).

0.2 0.4 0.8 1.6 3.2 6.25 12.5 25 50 100


Figure 3. Dose response of A431 cells to Epithelial growth factor (EGF) as measured by specific anti-body detecting phosphorylated ERK (Tyr204). The image represents a 96-well two color In Cell West-ern with the 800 and 700 channels detecting total and phosphorylated ERK, respectively. Background wells were incubated with secondary antibody but no primary antibody. The graph represents normal-ized quantitative data demonstrating the percent phosphorylation of ERK.

LI-COR Biosciences


Quantitative and simultaneous measurements of EGFR and ERK

phosphorylation in response to EGF stimulation.


images.


EGFR).

800 nm image(phosphorylated ERK).

0.2 0.4 0.8 1.6 3.2 6.25 12.5 25 50 100


Figure 4. Dose response of A431 cells to Epithelial growth factor (EGF) as measured by specific antibody detecting phosphory-lated EGF receptor (Tyr1045) and phosphorylated ERK (Tyr204) simultaneously. The image represents a 96-well two color In Cell Western with the 800 and 700 channels detecting phosphorylated ERK (Tyr204) and phosphorylated EGF receptor, respec-tively. Background wells were incubated with secondary antibody but no primary antibody.

Odyssey

®



Signal specificity confirmation of “In-Cell” Western using conventional

Western blots.

700 nm (red), 800 nm (green),and both (yellow).

700 nm image.

800 nm image.

Figure 5. Simultaneous measurement of total and phosphorylated EGF receptor in resting and EGF-treated A431 cell lyastes. Two-fold serials of dilutions of resting (lane 1 to 4) and EGF treated (lane 5 to 8) A431 cellular lysates were loaded, then the levels of phosphorylated EGFR (700 nm, red) and total EGFR (800 nm, green) in these lysates were simultaneously assessed.

1 2 3 4 5 6 7 8

LI-COR Biosciences


Simultaneous measurement of phosphorylated EGF receptor and total ERK

in resting and EGF-treated A431 cell lysates.


700 nm image.

800 nm image.

Figure 6. Two-fold serials of dilutions of resting (lane 1 to 4) and EGF treated (lane 5 to 8) A431 cellular lysates were loaded, then the levels of phosphorylated EGFR (700 nm, red) and total ERK (800 nm, green) in these lysates were simultaneously assessed.

1 2 3 4 5 6 7 8

Odyssey

®



Simultaneous measurement of total and phosphorylated ERK in resting and

EGF-treated A431 cell lysates.


700 nm image.

800 nm image.

Figure 7. Two-fold serials of dilutions of resting (lane 1 to 4) and EGF treated (lane 5 to 8) A431 cellular lysates were loaded, then the levels of phosphorylated ERK (700 nm, red) and total ERK (800 nm, green) in these lysates were simultaneously assessed.

1 2 3 4 5 6 7 8

LI-COR Biosciences


Simultaneous measurement of phosphorylated EGF receptor and ERK in

resting and EGF-treated A431 cell lysates.


700 nm image.

800 nm image.

Figure 8. Two-fold serials of dilutions of resting (lane 1 to 4) and EGF treated (lane 5 to 8) A431 cellular lysates were loaded, then the levels of phosphorylated EGFR (700 nm, red) and phosphorylated ERK (800 nm, green) in these lysates were simulta-neously assessed.

1 2 3 4 5 6 7 8

4308




www.licor.com

LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, inc. Alexa Fluor is a registered trademark of Invitrogen Corporation. Tween is a registered trademark of ICI Americas, Inc. Triton is a regis-tered trademark of Union Carbide Chemicals and Plastics Corp. Nunc and Microwell are trademarks of Nunc A/S Corporation. Falcon is a trade-mark of Becton Dickinson and Company. Gibco is a registered trademark of Invitrogen Corporation. The Odyssey Infrared Imaging System is covered by U.S. patent (6,495,812), foreign equivalents, and patents pending.

®

Odyssey


®


IRDye

®

800CW EGF Competition and Binding Assay Using A431 Cells

Revised September, 2006. The most recent version of this protocol is posted at http://biosupport.licor.com/support

®

Doc# 988-08625

Odyssey

®



Contents

Page


II. Seeding, Stimulation, and Detection with IRDye

®

800CW EGF.......................1




LI-COR Reagents

• IRDye

®

800CW EGF Optical Probe (Cat.# 926-08446)• Odyssey

®


Additional Reagents

• 1X PBS wash buffer.• Tissue culture reagents (serum, D-MEM, trypsin, 1X PBS).• TO-PRO

®

-3 was purchased from Molecular Probes.• 20% Tween

®

-20.• 37% formaldehyde.• 10% Triton

®

X-100.• Nunc™ 96 Microwell™ Plate ( Nunc, Cat.# 167008).• Primary antibodies as described below.

Special Note: Serum starvation of the cells is required to obtain maximal response.

II. Seeding, Stimulation and Detection with IRDye 800CW EGF

The following protocol is intended to illustrate the process for testing a particular cell type with IRDye 800CW EGF Optical Probe for eventual use

in vivo

. A431 (epithelial carcinoma) cells are used in this example, because of its over-expression of EGFR. Media considerations and certain cell characteristics will naturally alter this existing protocol.

1. Allow A431 cell growth in a T75 flask in D-MEM and 10% fetal calf serum (FCS; Gibco

®

) using standard tissue culture procedures until cells reach 80%-90% confluency (~1.5x10

7

cells).

2. Remove growth media, wash cells with sterile 1X PBS, and trypsinize cells.

3. Neutralize displaced cells with culture media and pellet by centrifugation.

LI-COR Biosciences


4. Remove supernatant and resuspend cell pellet in remaining media by manually tapping the collection tube. Avoid vigorous pipetting or vortexing to resuspend cells in order to maintain cell integrity.

5. Dilute cells to 20 ml in complete media and count cells using a hemacytometer.

6. Dilute cells with complete media to concentration of 200,000 cells/ml.

7. Gently mix the cell suspension thoroughly.

8. Under sterile conditions dispense 200 µl of the cell suspension per well in a Nunc™ 96 Microwell™ plate (40,000 cells plated per well).

9. Incubate cells and monitor cell density until cells are consistently confluent in each well. This should take approximately three days.


11. Remove complete media from the microwell plate by aspiration or inversion of the plate and tap excess media on tissue.

12. Replace media with 200 µl of pre-warmed serum-free media per well and incubate 4 to 16 hours.

13. In a separate 96-well Microwell™ plate devoid of cells, prepare a dilution series of reagents for the binding and competition assays discussed in step 14. Dispense 50 µl of D-MEM per well (exclude Wells 1, 2, and 12).

14. Continue preparation of the dilutions for both assays in the cell-less microtiter plate discussed above prior to adding to A431 cells.

Binding Assay

:

Prepare 1:2 serial dilutions of IRDye 800CW EGF ranging from 0.2 to 100 ng/ml. Dilute in D-MEM. Add 100 µl of IRDye 800CW EGF (100 ng/ml) to Well 12 in triplicate rows. Transfer 50 µl from Well 12 to Well 11 and mix by pipetting up and down. Repeat this process through to Well 3. Leave the first and second wells without EGF (background controls) and add 100 µl D-MEM only. An example image of the experimental layout should look like that shown in Figure 1A.

Competition Assay

:

Prepare 1:2 serial dilutions of unlabeled EGF (15 µg/ml; Dilute in D-MEM.) ranging from 15 to 0.03 µg/ml. Add 100 µl of 15 µg/ml unlabeled EGF to Well 12 in triplicate rows. Then transfer 50 µl from Well 12 to Well 11 and mix by pipetting up and down. Repeat this process to Well 3. Leave the first and second wells without EGF (background controls) and add 100 µl D-MEM only. Remove and discard 50 µl from Well 3 so all wells now contain 50 µl except Wells 1 and 2. Add 50 µl of 200 ng/ml IRDye 800CW EGF to Wells 3-12 for triplicate

Competition Assay

rows giving a final concentration of labeled EGF of 100 ng/ml. An example image of the experimental layout should look like that shown in Figure 1B.

15. Remove starvation media from cells by aspiration or inversion of the plate and tap excess media on tissue.

Do not store. Move directly to cell containing plate and begin.

Odyssey

®



16. Transfer 50 µl EGF dilutions for

Binding Assay

and 100 µl EGF mixtures for

Competition Assay

from the dilutions prepared in Step 14 into the cell-containing plate. Transfer these mixtures quickly (~20 sec) as the cellular responses are quick.

17. Incubate at room temperature for 2 minutes.

18. Prepare fresh

Fixing Solution

as follows:

1X PBS 45.0 ml37% Formaldehyde 5.0 ml

3.7% Formaldehyde 50.0 ml

19. Remove EGF-containing media by aspiration or inversion. Immediately fix cells by addition of 150 µl of fresh

Fixing Solution

and incubate at room temperature (RT) for 20 minutes with no shaking.

Add the

Fixing Solution

carefully by pipetting down the side of the wells to avoid detaching the cells.

Notes:

• Cover plate with foil to protect from light.

20. Prepare


as follows:


1X PBS + 0.1% Triton X-100 500 ml

21. Remove the

Fixing Solution

by aspiration.

22. Wash four times with 200 µl of


for 5 minutes per wash to permeabilize the cells.

Notes:

• Allow each wash to shake on a rotator for 5 minutes at RT.

• Do not allow cells/wells to become dry during washing. Add washes immediately after each other.

• Cover plate to protect from light during procedure washings.

23. Remove the


by aspiration or inversion.

24. To each well, carefully add 150 µl of LI-COR Odyssey Blocking Buffer (#927-40000) + 0.1% Tween-20 down the side of the wells and incubate for 1.5 hours at RT with moderate shaking on a rotating platform.

25. IRDye 800CW EGF will be detected in the 800-channel. TO-PRO-3 is a cell stain that can be used to normalize the signal from binding of the labeled EGF, to correct for variations in cell number from well to well. Dilute the stain 1:5000 in LI-COR Odyssey Blocking Buffer. Proceed to Step 26.

26. Add 50 µl of LI-COR Odyssey Blocking Buffer + 0.1% Tween-20 to Wells 1 and 2. These wells will serve as controls for any potential background due to the stain.

27. Dilute TO-PRO-3 (1:5,000 dilution; Molecular Probes Cat# T-3605) in Odyssey blocking buffer. Add 50 µL to each well (except Well 1) and incubate for 1 hour with gentle shaking.

Avoid prolonged light exposure of the antibody and stain vials.

LI-COR Biosciences






• In Cell Western assays require sterile plates for tissue culture growth. The following plates are recommended by LI-COR Biosciences:




• Plates deviating from LI-COR recommendations may require lower or higher focus offsets for optimal resolu-tion and detection. If alternative plates are used, an initial optimization scan will be necessary. Scan a plate containing experimental and control samples at 0.5, 1.0, 2.0, 3.0, and 4.0 mm focus offsets. Use the same

28. Prepare


as follows:





(step 28) by gently adding buffer down the side of the wells to avoid detaching the cells. Use a generous amount of buffer (200 µl). Allow wash to shake gently on a rotator for 5 minutes at RT.



32. Before scanning, clean the bottom plate surface and the Odyssey Imager scanning surface with moist, lint-free paper to avoid obstructions during scanning.

33. Scan the plate simultaneously at 700 nm and 800 nm using the Odyssey Infrared Imaging System. Use medium scan quality, 169 µm resolution, 3.0 mm focus offset, and an intensity setting of 5 for the 700 channel and 7 for the 800 nm channel.


Odyssey

®



intensity settings for each scan. After reviewing the collected scans, use the focus offset with the highest sig-nal-to-noise as your focus offset for experiments using the alternate plates.


• Start with an intensity of 5 for the 700 nm channel and 7 for the 800 nm channel. If your image signal is satu-rated or too high, re-scan using a lower intensity setting (i.e., 2.5 (700 nm) or 5 (800 nm)). If your image sig-nal is too low, re-scan using a higher intensity setting (i.e., 7.5 (700 nm) or 8 (800 nm)).



Note:

The level of blocking achieved on any given cell line will be a helpful indicator of how effective the probe will be

in vivo

.

0

0.5

1.5

2.5

3.5

0 1 10 100

nM

Rel

ativ

e In

ten

sity

x 1

04

(A)

1.0 2.0 3.9 7.8 15.6 31 63 125 250 500 ng/ml IRDye 800CW EGF

Figure 1. Plate set up for binding (A) and competition (B) assays. Subsequent analyses are shown on the right. Normalization using TO-PRO-3 is detected in the 700-channel while IRDye 800CW EGF binding (A) and IRDye 800CW EGF blocking with increasing concentrations of unlabeled EGF is depicted in (B).

(B)

0Bkg

0.03

100

0.05

100

0.1

100

0.2

100

0.5

100

0.9

100

1.9

100

3.8

100

7.5

100

15

100

µg/ml Unlabeled EGF

ng/ml IRDye 800CW EGF

0

0

Bkg

Bkg

0

20

40

60

80

100

120

0.00 0.01 0.10 1.00 10.00

% M

axim

um

Inte

nsi

ty

[unlabeled EGF] µM

4647 Superior Street

• P.O. Box 4000 • Lincoln, Nebraska 68504 USATechnical Support: 800-645-4260



www.licor.com

LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, inc. TO-PRO is a registered trademark of Invitrogen Corporation. Tween is a registered trademark of ICI Americas, Inc. Triton is a registered trademark of Union Carbide Chemicals and Plastics Corp. Nunc and Microwell are trademarks of Nunc A/S Corporation. Falcon is a trademark of Becton Dickinson and Company. Gibco is a registered trademark of Invitrogen Corporation. All other trademarks belong to their respective owners. The Odyssey Infrared Imaging System is covered by U.S. patents, foreign equivalents, and patents pending.

®

Odyssey


®


Complete Sample Protocol for Measuring IC

50

of Inhibitor PD168393 in A431 Cells Responding to Epidermal Growth Factor

Revised September, 2006. The most recent version of this protocol is posted at http://biosupport.licor.com/support

®

Doc# 988-08599

Odyssey

®



Contents

Page


II. Sample Protocol ...............................................................................................2




Odyssey

®

Reagents

• IRDye™ 800CW- and IRDye™ 680-labeled secondary antibodies (LI-COR)*• Odyssey

®


Additional Reagents

• 1X PBS wash buffer• Tissue culture reagents (serum, D-MEM, trypsin, 1X PBS)• 20% Tween

®

-20• Epidermal Growth Factor (Upstate Group Inc., Cat.# 01-107)• Protein Tyrosine Kinase Inhibitor PD168393 (CALBIOCHEM

®

, Cat.# 513033)• 37% formaldehyde• 10% Triton

®

X-100• Falcon 384-well microplate (Cat.# 353961)• Primary antibodies

Special Note: Anti-phosphorylated-EGFR and anti-phosphorylated-ERK antibodies are purchased from Cell Signaling Technology and Santa Cruz Biotechnology, respectively. Cell starvation is needed to obtain maximal response when these two phospho-antibodies are used. This is in contrast to use of anti-phospho-ERK from BD Pharmingen and from Cell Signaling Technology.

* IRDye™ 800CW-labeled secondary antibodies are also available from Rockland Immunochemicals, Inc. Alexa Fluor

®


LI-COR Biosciences


II. Sample Protocol

1. Allow A431cell growth in a T75 flask using standard tissue culture procedures until cells reach near confluency (~1.5x10

7

cells; D-MEM, 10% FBS; Gibco

®

).

2. Remove growth media, wash cells with sterile 1X PBS, and trypsinize cells for displacement.

3. Neutralize displaced cells with culture media and clarify by centrifugation.

4. Remove supernatant and disrupt the cell pellet manually by hand tapping the collection tube. Avoid use of pipet or vortex during pellet disruption to maintain cell integrity.

5. Resuspend cells in 20 ml of complete media and count cells using a hemacytometer.

6. Dilute cells with complete media such that 200,000 cells/ml is achieved.

7. Manually mix the cell suspension thoroughly.

8. Under sterile conditions dispense 50 µl of the cell suspension per well in Falcon 384-well microplate (10,000 cells plated per well).

9. Incubate cells and monitor cell density until confluency is achieved with well-to-well consistency; approximately three days.

10. Warm serum-free media (D-MEM; Gibco) to 37 °C.

11. Remove complete media from plate wells by aspiration or manual displacement.

12. Replace media with 50 µl of pre-warmed serum free media per well and incubate 4 to 16 hours.


14. Dissolve PD168393 in D-MEM to make 3 µM stock. Make two fold serial dilutions of inhibitor using D-MEM so that the final concentration of inhibitor range from 3 µM to 90 pM, as shown in section


.

15. Remove media in A431 cell plate.

16. Add 50 µl of serial diluted inhibitor into cells and incubate 1 to 2 hours.

17. Remove inhibitor from plate wells by aspiration or manual displacement.

18. Add either serum free media for resting cells (mock) or serum free media with 100 ng/ml EGF. Use 50 µl of resting/activation media per well.

19. Allow incubation at 37 °C for 7.5 minutes.

Odyssey

®



20. Remove activation or stimulation media manually or by aspiration. Immediately fix cells with 4% formaldehyde in 1X PBS for 20 minutes at room temperature.

a. Prepare fresh

Fixing Solution

as follows:

1X PBS 45 ml37% Formaldehyde 5 ml

3.7% Formaldehyde 50 ml

b. Using a multi-channel pipettor, add 150 µl of fresh

Fixing Solution

(room temperature solution, RT).

Add the

Fixing Solution

carefully by pipetting down the sides of the wells to avoid detaching the cells from the well bottom.

c. Allow incubation on bench top for 20 minutes at RT with no shaking.

21. Wash five times with 1X PBS containing 0.1% Triton X-100 (cell permeabilization) for 5 minutes per wash.

a. Prepare


as follows:


1X PBS + 0.1% Triton X-100 500 ml

b. Remove

Fixing Solution

to an appropriate waste container (contains formaldehyde).

c. Using a multi-channel pipettor, add 200 µl of


(RT).

Make sure to carefully add the solution down the sides of the wells to avoid detaching the cells.

d. Allow wash to shake on a rotator for 5 minutes at RT.

e. Repeat washing steps 4 more times after removing wash manually.

Do not allow cells/wells to become dry during washing. Immediately add the next wash after manual disposal.

LI-COR Biosciences


22. Using a multi-channel pipettor, block cells/wells by adding 50 µl of LI-COR Odyssey Blocking Buffer to each well.

Add the solution carefully by pipetting down the sides of the wells to avoid detaching the cells.

Notes:

• No single blocking reagent will be optimal for every antigen-antibody pair. Some primary antibodies may exhibit greatly reduced signal or different nonspecific banding in different blocking solutions. If you have dif-ficulty detecting your target protein, changing the blocking solution may dramatically improve performance. If the primary antibody has worked well in the past using chemiluminescent detection, try that blocking solu-tion for Aerius detection.




23. Allow blocking for 90 minutes at RT with moderate shaking on a rotator.

24. Add the two primary antibodies into a tube containing Odyssey Blocking Buffer. Choose one of the following primary antibody pairs:

• Phospho-ERK (mouse; 1:100 dilution; Santa Cruz Biotechnology, SC-7383) Total ERK1 (Rabbit; 1:200 dilution; Santa Cruz Biotechnology, SC-94)

• Phospho-EGFR Tyr1045 (Rabbit; 1:100 dilution; Cell Signaling Technology, 2237)Total ERK2 (Mouse; 1:75 dilution; Santa Cruz Biotechnology, SC-1647)

• Phospho-EGFR Tyr1045 (Rabbit; 1:100 dilution; Cell Signaling Technology, 2237)Phospho-ERK (Mouse; 1:100 dilution; Santa Cruz Biotechnology, SC-7383)

• Phospho-EGFR Tyr1045 (Rabbit; 1:100 dilution; Cell Signaling Technology, 2237)Total EGFR (Mouse; 1:500 dilution; Biosource International, AHR5062)

a. Mix the primary antibody solution well before addition to wells.

b. Remove blocking buffer from the blocking step and add 20 µl of the desired primary antibody or anti-bodies in Odyssey Blocking Buffer to cover the bottom of each well.

c.

Make sure to include control wells without primary antibody to serve as a source for background well intensity

. Add 50 µl of Odyssey Blocking Buffer only to control wells.

25. Incubate with primary antibody overnight with gentle shaking at RT.

Odyssey

®



26. Wash the plate five times with 1x PBS + 0.1% Tween-20 for 5 minutes at RT with gentle shaking, using a generous amount of buffer.

a. Prepare


as follows:



b. Using a multi-channel pipettor add 200 µl of


(RT).

Make sure to carefully add solution down the sides of the wells to avoid detaching the cells from the well bottom.

c. Allow wash to shake on a rotator for 5 minutes at RT.

d. Repeat washing steps 4 more times.

27. Dilute the fluorescently labeled secondary antibody in Odyssey Blocking Buffer as specified below. To lower background, add Tween-20 to the diluted antibody for a final concentration of 0.2%.

Goat anti-rabbit IRDye™ 680 (1:200 dilution; LI-COR Cat.# 926-32221)Goat anti-mouse IRDye™ 800CW (1:800 dilution; LI-COR Cat.# 926-32210)

Or

Goat anti-mouse IRDye™ 680 (1:200 dilution; LI-COR Cat.# 926-32220)Goat anti-rabbit IRDye™ 800CW (1:800 dilution; LI-COR Cat.# 926-32211)



Notes:

• Use IRDye™ 800CW-labeled secondary antibody to detect phosphorylation and IRDye™ 680-labeled sec-ondary antibody to detect total protein.

28. Mix the antibody solutions well and add 20 µl of the secondary antibody solution to each well. Incubate for 60 minutes with gentle shaking at RT. Protect plate from light during incubation.


a. Using a multi-channel pipettor, add 200 µl of


at RT (see step 26).

Make sure to carefully add solution down the sides of the wells to avoid detaching the cells from the well bottom

.

b. Allow wash to shake on a rotator for 5 minutes at RT.

c. Repeat washing steps 4 more times after removing wash manually.



31. Before plate scanning, clean the bottom plate surface and the Odyssey Imager scanning bed with moist lint free paper to avoid any obstructions during scanning.

LI-COR Biosciences






• In-Cell Western assays require sterile plates for tissue culture growth. The following 96- and 382-well plates are recommended by LI-COR Biosciences:




• If you use plates other than the plates recommended above, the focus offset can be determined by scanning a plate containing experimental and control samples at 0.5, 1.0, 2.0, 3.0, and 4.0 mm focus offsets. Use the same intensity settings for each scan. After reviewing the collected scans, use the focus offset with the highest signal-to-noise as your focus offset for experiments.




• Establish the specificity of your primary antibody by screening lysates through Western blotting and detection on the Odyssey instrument. If significant non-specific banding is present, choose alternative primary antibod-ies. Non-specific binding of primaries will complicate interpretation of In-Cell Western results.

32. Scan the plate with detection in both the 700 and 800 channels using the Odyssey Infrared Imaging System (700 nm detection for IRDye™ 680 antibody and 800 nm detection for IRDye™ 800CW antibody). Use medium quality, 169 µm resolution, 3.0 mm focus offset, and an intensity setting of 5 for both 700 and 800 nm channels.

Odyssey

®




Simultaneous measurement of the effect of PD168393 on the

phosphorylation of EGFR and downstream ERK.

Four different experiments were conducted on the same microplate. (Color images can be seen at http://biosupport.licor.com/support.)

Experiment 1. Effect on the phosphorylation of EGFR (normalized against total EGFR).

Two-color display of both 700 and 800 nm channels.

700 nm image (phosphorylated EGFR).

800 nm image (total EGFR).

The level of EGFR phosphorylation was assessed in the 700 nm channel. A dramatic increase of EGFR phosphorylation in response to EGF stimulation in the 7th and 8th wells was seen, compared to the basal level of phosphorylation in the 5th and 6th wells (without EGF). Dose-dependent inhibition of EGFR phosphorylation by PD168393 was observed in the 9th to 24th wells. Similar amounts of EGFR are present in all wells, as indicated by the 800 channel. The 1st to 4th wells, reacting only with the secondary antibodies, serve as a negative control and background subtraction. R = resting cells; A = activated cells.

R R A A R R A A A A A A A A A A A A A A A A A A

- - - - ++

1° AB2° AB

EGF - - - - - - - - 0.09 0.19 0.38 0.75 1.5 3 6 12 24 47 94 188 375 750 1500 3000nM Inhibitor

duplicate rows

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Well

LI-COR Biosciences


Experiment 2. Effect on the phosphorylation of EGFR (normalized against total ERK).



800 nm image (total ERK).

The level of EGFR phosphorylation was assessed in the 700 nm channel. A dramatic increase of EGFR phosphorylation in response to EGF stimulation in the 7th and 8th wells was seen, compared to the basal level of phosphorylation in the 5th and 6th wells (without EGF). Dose-dependent inhibition of EGFR phosphorylation by PD168393 was observed in the 9th to 24th wells. Similar amounts of ERK are present in all wells, as indicated by the 800 chan-nel. The 1st to 4th wells reacting only with the sec-ondary antibodies, serve as a negative control and background subtraction. R = resting cells; A = activated cells.


- - - - ++

1° AB2° AB

EGF - - - - - - - - 0.09 0.19 0.38 0.75 1.5 3 6 12 24 47 94 188 375 750 1500 3000nM Inhibitor

duplicate rows

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Well

Odyssey

®



Experiment 3. Effect on the phosphorylation of ERK (normalized against total ERK).


700 nm image (phosphorylated ERK).


The level of ERK phosphorylation was assessed in the 700 nm channel. A dramatic increase of ERK phosphorylation in response to EGF stimulation in the 7th and 8th wells was seen, compared to the basal level of phosphorylation in the 5th and 6th wells (without EGF). Dose-dependent inhibition of ERK phosphorylation by PD168393 was observed in the 9th to 24th wells. Similar amounts of ERK are present in all wells, as indicated by the 800 chan-nel. The 1st to 4th wells reacting only with the sec-ondary antibodies, serve as a negative control and background subtraction. R = resting cells; A = acti-vated cells.


- - - - ++

1° AB2° AB

EGF - - - - - - - - 0.09 0.19 0.38 0.75 1.5 3 6 12 24 47 94 188 375 750 1500 3000nm Inhibitor

duplicate rows

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Well

LI-COR Biosciences


Experiment 4. Effect on the phosphorylation of EGFR and ERK.




R

The degree of phosphorylation of EGFR and ERK was assessed in the 700 nm (red) and 800 nm (green) channels, respectively. A dramatic increase of phosphorylation of EGFR and ERK in response to EGF stimulation in the 7th and 8th wells was seen, compared to the basal level phosphorylation in the 5th and 6th wells (without EGF). Dose-dependent inhibition of phosphorylation of EGFR and ERK by PD168393 was observed in 9th to 24th wells. The 1st to 4th wells reacting only with the secondary antibodies, serve as a negative control and background sub-traction. R = resting cells; A = activated cells.

R A A R R A A A A A A A A A A A A A A A A A A

- - - - ++

1° AB2° AB

EGF - - - - - - - - 0.09 0.19 0.38 0.75 1.5 3 6 12 24 47 94 188 375 750 1500 3000nM Inhibitor

duplicate rows

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Well





www.licor.com

LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, inc. Alexa Fluor is a registered trademark of Invitrogen Corporation. Tween is a registered trademark of ICI Americas, Inc. Triton is a regis-tered trademark of Union Carbide Chemicals and Plastics Corp. CALBIOCHEM is a registered trademark of EMD Biosciences Inc. Nunc and Microwell are trademarks of Nunc A/S Corporation. Falcon is a trademark of Becton Dickinson and Company. Gibco is a registered trademark of Invitrogen Corporation. The Odyssey Infrared Imager and the Odyssey system are covered by U.S. patent (6,495,812), foreign equivalents, and patents pending.

®

Odyssey


®


Complete Sample Protocol for Measuring IC

50

of Inhibitor U0126 in NIH3T3 Responding to Acidic Fibroblast Growth Factor (aFGF-1)


®

Doc# 988-08334

Odyssey

®



Contents

Page






Odyssey

®

Reagents

• IRDye™ 800CW-labeled goat anti-mouse secondary antibodies (LI-COR, Cat.# 926-32210)*• IRDye™ 680-labeled goat anti-rabbit secondary antibodies (LI-COR, Cat.# 926-32221)*• Odyssey

®

Blocking Buffer (Cat.# 927-40000)

Additional Reagents

• 1X PBS wash buffer• Tissue culture reagents (serum, D-MEM, trypsin, 1X PBS)• SIGMAScreen™ Poly-D-Lysine coated 96-well microplate (Sigma

®

, Cat.# Z38249-3)• Heparin (CALBIOCHEM

®

, Cat.# 375097)• Acidic Fibroblast Growth Factor (Upstate Group Inc., Cat.# 01-116)• MEK inhibitor U0126 (Promega

®

, Cat.# V1121)• Primary antibodies• 20% Tween

®

-20• 37% formaldehyde• 10% Triton

®

X-100

Special Note:

NIH3T3 cells do not adhere strongly to TC treated plates resulting in the need for poly-D-lysine coated plates in this assay. However, even with lysine coated plates the adherence of cells remains relatively weak compared to other cell lines.

Be very cautious and delicate with plate handling and pipetting when washing, removing, and add-ing solutions to avoid detaching the cells.


®


LI-COR Biosciences


II. Sample Protocol

1. Allow NIH3T3 (ATCC; CRL-1555) cell growth in a T75 flask using standard tissue culture procedures until cells reach near confluency (~1.5x10

7


®

).


3. Neutralize displaced cells with culture media and clarify by centrifugation (500 x g).

4. Remove supernatant and disrupt the cell pellet manually by hand tapping the collection tube. Avoid employment of pipet or vortex during pellet disruption to maintain cell integrity.


6. Reconstitute cells and dilute in 40 ml of complete media such that 75,000 cells/ml is achieved (2 plates x 96 wells x 200 µl/well = ~ 40 ml).


8. Under sterile conditions, dispense 200 µl of the cell suspension per well into a SIGMAScreen™ Poly-D-Lysine 96-well microplate (15,000 cells plated per well).

9. Incubate cells and monitor cell density until 70% confluency is achieved (it takes about 24 hours).

70% confluency is very important. 90 to 100% confluent cells are certain to detach during washing.


11. Dissolve U0126 in DMSO to make 10 mM stock. Make two fold serial dilutions of inhibitor using D-MEM. Add 10 µl of serial diluted inhibitor into cells so that the final concentration of inhibitor range from 1 to 125 µM (see Figure 1, section


). Incubate 1 to 2 hours.

12. Remove media and inhibitor from plate wells by aspiration or manual displacement.

13. Add either serum free media for resting cells (mock) or serum free media with 100 ng/ml aFGF combined with 10 µg/ml heparin for activated cells. Use 100 µl of resting/activation media per well.



a. Prepare fresh

Fixing Solution

as follows:




Fixing Solution


Make sure to carefully add the solution down the sides of the wells to avoid detaching the cells from the well bottom.


Odyssey

®




a. Prepare


as follows:


1X PBS + 0.1% Triton X-100 500 ml

b. Remove

Fixing Solution




(RT). Make sure to carefully add the solution down the sides of the wells to avoid detaching the cells from the well bottom.






Notes:






LI-COR Biosciences


19. Add the two primary antibodies to a tube containing Odyssey Blocking Buffer. Combine the solutions defined below for ERK target analysis:

• Phospho-ERK (Rabbit; 1:100 dilution; Cell Signaling Technology 9101) Total ERK2 (Mouse; 1:75 dilution; Santa Cruz Biotechnology SC-1647)


b. Remove blocking buffer from the blocking step and add 50 µl of the desired primary antibody or antibodies in Odyssey Blocking Buffer to cover the bottom of each well.

c.





a. Prepare


as follows:



b. Using a multi-channel pipettor, add 200 µl of


(RT).




22. Dilute the fluorescently labeled secondary antibody in Odyssey Blocking Buffer as specified below. To lower background, add Tween-20 to the diluted antibody to a final concentration of 0.2%.

Goat anti-rabbit IRDye™ 680 (1:200 dilution; LI-COR)Goat anti-mouse IRDye™ 800CW (1:800 dilution; LI-COR)




24. Wash the plate five times with 1X PBS + 0.1% Tween-20 for 5 minutes at RT with gentle shaking, using a generous amount of buffer.








Odyssey

®




• The Odyssey Imager requires that microplates have a maximum 4.0 mm distance from the Odyssey scanning surface to the target detection area of the plate. The recommended focus offset is 3.0 mm for the SigmaScreen™ microplates specified for this assay.

• If you use plates other than the recommended SigmaScreen™ microplates, the focus offset can be determined by scanning a plate containing experimental and control samples at 0.5, 1.0, 2.0, 3.0, and 4.0 mm focus off-sets. Use the same intensity settings for each scan. After reviewing the collected scans, use the focus offset with the highest signal-to-noise as your focus offset for experiments.




• Establish the specificity of your primary antibody by screening lysates through Western blotting and detection on the Odyssey instrument. If significant non-specific banding is present, choose alternative primary antibod-ies. Non-specific binding of primaries will complicate interpretation of In-Cell Western assay results.



26. Scan the plate with detection in both the 700 and 800 channels using the Odyssey instrument (700 nm detection for IRDye™ 680 antibody and 800 nm detection for IRDye™ 800CW antibody). Use medium scan quality, 169 µm resolution, 3.0 mm focus offset, and an intensity setting of 5 for both 700 and 800 nm channels

LI-COR Biosciences



Color images can be seen at http://biosupport.licor.com/support.


700 nm image (phospho-ERK).


100 100 100 100 100 100 100 100 100 ng/ml FGF0 1 2 4 8 16 32 62.5 125 um U0126

Background Resting

Figure 1. U0126 inhibition of ERK phosphorylation in NIH3T3 cells stimulated with FGF. The graph demonstrates the inhibitory effect of the MEK inhibitor U0126 as determined through the detec-tion of ERK phosphorylation (Thr202/Tyr204) within an ICW assay. Resulting data were plotted and the IC50 of U0126 was determined to be ~15µM, correlating well with the IC50 reported in literature (1) for in vitro and in vivo assays.

1. Ahn, N.G. et al (1999). U0126: An Inhibitor of MKK/ERK Signal Transduction in Mammalian Cells. Promega Notes 71, p. 4.

4308




www.licor.com

LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, inc. Alexa Fluor and Gibco are registered trademarks of Invitrogen Corporation. Tween is a registered trademark of ICI Americas, Inc. Triton is a registered trademark of Union Carbide Chemicals and Plastics Corp. Promega is a registered trademark of Promega Corporation. Sigma and SIGMAScreen are trademarks or registered trademarks of Sigma-Aldrich Inc. CALBIOCHEM is a registered trademark of EMD Biosciences Inc. The Odyssey Infrared Imaging System is covered by U.S. patent (6,495,812), foreign equivalents, and patents pending.

®

Odyssey


®


Complete Sample Protocol Detailing the Seeding, Stimulation, and Detection of the HeLa Cellular Response to Epidermal Growth Factor


®

Doc# 988-08335

Odyssey

®



Contents

Page






Odyssey

®

Reagents

• IRDye™ 800CW-labeled goat anti-mouse secondary antibodies (LI-COR, Cat.# 926-32210)*• IRDye™ 680-labeled goat anti-rabbit secondary antibodies (LI-COR, Cat.# 926-32221)*• Odyssey

®

Blocking Buffer (927-40000)

Additional Reagents


®

-20• Epidermal Growth Factor (Upstate Group Inc., 01-107)• 37% formaldehyde• 10% Triton

®

X-100• Nunc™ 96 Microwell™ Plate (Nunc, 167008)• Primary antibodies

Special Note: (1) long passage HeLa cells are not suitable for the assay because of high basal level phosphorylation of ERK. As a result, ordering a new sample of this cell line from ATCC is highly recom-mended. (2) No starvation is needed or recommended.


®


LI-COR Biosciences


II. Sample Protocol

1. Allow HeLa (ATCC; CCL-2) cell growth in a T75 flask using standard tissue culture procedures until cells reach near confluency (~1.5x10

7


®

).





6. Dilute cells with complete media such that 75,000 cells/ml is achieved.


8. Under sterile conditions, dispense 200 µl of the cell suspension per well in a Nunc™ 96 Microwell™ Plate (15,000 cells plated per well).

9. Incubate cells and monitor cell density until confluency is achieved with well-to-well consistency; approximately two to three days.

10. Warm serum-free media (D-MEM; Gibco) to 37 °C. Dispense 100 µl of D-MEM per well in the Nunc 96-well microplate.

11. Make serial dilutions of EGF in the microplate ranging from 0.2 to 100 ng/ml. Leave the first and second wells without EGF (resting cells as control), as shown in section


.


13. Transfer media from the dilution plate into the experimental plate.



a. Prepare fresh

Fixing Solution

as follows:




Fixing Solution


Add the

Fixing Solution



Odyssey

®




a. Prepare


as follows:


1X PBS + 0.1% Triton X-100 500 ml

b. Remove

Fixing Solution




(RT).







Notes:






LI-COR Biosciences


19. Add the two primary antibodies to a tube containing Odyssey Blocking Buffer. Combine the following solutions as defined below for ERK target analysis:

• Phospho-ERK (Rabbit; 1:100 dilution; Cell Signaling Technology 9101)

• Total ERK2 (Mouse; 1:75 dilution; Santa Cruz Biotechnology SC-1647)



c.


(see first well in Figure 1). Add 50 µl of Odyssey Blocking Buffer only to control wells.


Notes:



a. Prepare


as follows:





(RT).





Goat anti-rabbit IRDye™ 680 (1:200 dilution; LI-COR)Goat anti-mouse IRDye™ 800CW (1:800 dilution; LI-COR)







at RT (see step 21). Make sure to carefully add solution down the sides of the wells to avoid detaching the cells from the well bottom.




Odyssey

®







• In-Cell Western assays require sterile plates for tissue culture growth. The following plates are recommended by LI-COR Biosciences:




• If you use plates other than those recommended above, the focus offset can be determined by scanning a plate containing experimental and control samples at 0.5, 1.0, 2.0, 3.0, and 4.0 mm focus offsets. Use the same intensity settings for each scan. After reviewing the scans, use the focus offset with the highest signal-to-noise for your experiments.





26. Before plate scanning, clean the bottom plate surface and the Odyssey scanning bed with moist lint free paper to avoid any obstructions during scanning.


LI-COR Biosciences








0.2 0.4 0.8 1.6 3.2 6.25 12.5 25 100ng/ml

Background Resting

Figure 1. Dose response of HeLa cells to epider-mal growth factor (EGF) as measured by specific antibody detecting dual-phosphorylated ERK (Thr202/Tyr204). The image represents a 96-well two color In-Cell Western with the 800 and 700 channels detecting total and phosphorylated ERK, respectively. Background wells were incubated with secondary antibody but no primary antibody. The graph represents normalized quantitative data demonstrating the percent phosphorylation of ERK.

50

duplicate rows

4308




www.licor.com

LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, inc. Alexa Fluor and Gibco are registered trademarks of Invitrogen Corporation. Tween is a registered trademark of ICI Americas, Inc. Triton is a registered trademark of Union Carbide Chemicals and Plastics Corp. Sigma and SIGMAScreen are trademarks or registered trademarks of Sigma-Aldrich Inc. Nunc and Microwell are trademarks of Nunc A/S Corporation. Falcon is a trademark of Becton Dickinson and Company. The Odyssey Infrared Imaging System is covered by U.S. patent (6,495,812), foreign equivalents, and patents pending.

®

Odyssey


®


Complete Sample Protocol Detailing the Seeding, Stimulation, and Detection of the NIH3T3 Cellular Response to Platelet Derived Growth Factor BB (PDGF-BB)


®

Doc# 988-08336

Odyssey

®



Contents

Page






Odyssey

®

Reagents


®


Additional Reagents


®

, Cat.# Z38249-3)• Platelet Derived Growth Factor BB (PDGF-BB) (Upstate Group Inc., Cat.# 01-305)• Primary antibodies• 20% Tween

®


®

X-100

Special Note:




®


LI-COR Biosciences


II. Sample Protocol


7


®

).












12. Add either serum free media for resting cells (mock) or serum free media with serial concentrations of PDGF-BB ranging from 0.4 to 200 ng/ml for activated cells. Use 100 µl of resting/activation media per well.



a. Prepare fresh

Fixing Solution

as follows:




Fixing Solution




Odyssey

®




a. Prepare


as follows:


1X PBS + 0.1% Triton X-100 500 ml

b. Remove

Fixing Solution




(RT).







Notes:






LI-COR Biosciences


18. Add the two primary antibodies into a tube containing Odyssey Blocking Buffer. Combine the solutions defined below for phospho-Akt target analysis, using total ERK2 for normalization:

• Phospho-Akt (Rabbit; 1:100 dilution; Cell Signaling Technology 9271)

• Total ERK2 (Mouse; 1:100 dilution; Santa Cruz Biotechnology SC-1647)



c.





a. Prepare


as follows:





(RT).





Goat anti-rabbit IRDye™ 800CW (1:800 dilution; LI-COR, Cat.#926-32211)Goat anti-mouse IRDye™ 680 (1:200 dilution; LI-COR, Cat.#926-32220)







at RT (see step 20). Make sure to carefully add the solution down the sides of the wells to avoid detaching the cells from the well bottom.




Odyssey

®











26. Before plate scanning, clean the bottom plate surface and the Odyssey Imager scanning surface with moist lint free paper to avoid any obstructions during scanning.


LI-COR Biosciences





200 n

g/ml

Figure 1. Dose response of NIH3T3 cells to Platelet Derived Growth Factor (PDGF-BB) as measured by specific antibody detecting phosphorylated Akt (Ser473) using total ERK2 for normalization. The image represents a 96-well two-color In-Cell Western with the 700 and 800 channels detecting total ERK2 and phosphorylated Akt (Ser473) respectively. Background wells were incubated with secondary antibody but no primary antibody. The graph represents average of four sets quantitative data demonstrating the percent induction of phosphorylated Akt (Ser473).

100 n

g/ml

50 n

g/ml

12.5

ng/ml

6.3 n

g/ml

3.1 n

g/ml

1.6 n

g/ml

0.8 n

g/ml

0.4 n

g/ml

0 ng/m

l

25 n

g/ml

Backg

round

700 nm channel image (total Erk for normalization).

800 nm channel image (phospho-Akt).

4308




www.licor.com

LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, inc. Tween is a registered trademark of ICI Americas, Inc. Triton is a registered trademark of Union Carbide Chemicals and Plastics Corp. Sigma and SIGMAScreen are trademarks or registered trademarks of Sigma-Aldrich Inc. Calbiochem is a registered trademark of EMD Biosciences Inc. Gibco and Alexa Fluor are registered trademarks of Invitrogen Corporation. The Odyssey Infrared Imaging System is covered by U.S. patent (6,495,812), foreign equivalents, and patents pending.

®

Odyssey


®


Complete Sample Protocol Detailing the Seeding, Stimulation, and Detection of the NIH3T3 Cellular Response to Acidic Fibroblast Growth Factor (aFGF-1)


®

Doc# 988-08337

Odyssey

®



Contents

Page






Odyssey

®

Reagents


®


Additional Reagents


®

, Cat.# Z38249-3)• Heparin (CALBIOCHEM

®

, 375097)• Acidic Fibroblast Growth Factor (Upstate Group Inc., Cat.# 01-116)• Primary antibodies• 20% Tween

®


®

X-100

Special Note:




®


LI-COR Biosciences


II. Sample Protocol


7


®

).












12. Add either serum free media for resting cells (mock) or serum free media with serial concentrations of aFGF-1 ranging from 0.2 to 100 ng/ml, combined with 10 µg/ml heparin for activated cells. Use 100 µl of resting/activation media per well.



a. Prepare fresh

Fixing Solution

as follows:




Fixing Solution




Odyssey

®




a. Prepare


as follows:


1X PBS + 0.1% Triton X-100 500 ml

b. Remove

Fixing Solution




(RT).

Make sure to carefully add the solution down the sides of the wells to avoid detaching the cells from the well bottom

.





Add the solution carefully by pipetting down the sides of the wells to avoid detaching the cells

.

Notes:






LI-COR Biosciences


18. Add the two primary antibodies to a tube containing Odyssey Blocking Buffer. Combine the solutions defined below for phospho-ERK target analysis, using total ERK2 for normalization:

• Phospho-ERK (Rabbit; 1:100 dilution; Cell Signaling Technology Cat.# 9101)

• Total ERK2 (Mouse; 1:100 dilution; Santa Cruz Biotechnology Cat.# SC-1647)



c.





a. Prepare


as follows:





(RT).


.




Goat anti-rabbit IRDye™ 680 (1:200 dilution; LI-COR Cat.# 926-32221)Goat anti-mouse IRDye™ 800CW (1:800 dilution; LI-COR Cat.# 926-32210)









.




Odyssey

®













LI-COR Biosciences





Backg

round

Figure 1. Dose response of NIH3T3 cells to Fibroblast growth factor (aFGF-1) as measured by specific antibody detecting dual-phosphorylated ERK (Thr202/Tyr204). The image represents a 96-well two-color In-Cell Western with the 800 and 700 channels detecting total and phosphorylated ERK respectively. Background wells were incubated with secondary antibody, but no primary antibody. The graph repre-sents normalized quantitative data demonstrating the percent phosphorylation of ERK.

100 n

g/ml

50 n

g/ml

12.5

ng/ml

6.3 n

g/ml

3.1 n

g/ml

1.6 n

g/ml

0.8 n

g/ml

0.4 n

g/ml

0.2 n

g/ml

25 n

g/ml

Restin

g

duplicate rows

4308




www.licor.com

LI-COR is an ISO 9001 registered company. © 2004 LI-COR Inc. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, inc. Tween is a registered trademark of ICI Americas, Inc. Triton is a registered trademark of Union Carbide Chemicals and Plastics Corp. Sigma and SIGMAScreen are trademarks or registered trademarks of Sigma-Aldrich Inc. CALBIOCHEM is a registered trademark of EMD Bio-sciences Inc. Gibco and Alexa Fluor are registered trademarks of Invitrogen Corporation. The Odyssey Infrared Imaging System is covered by U.S. patent (6,495,812), foreign equivalents, and patents pending.

®

Odyssey


®


Complete Apoptosis Assay Example Detailing the Seeding, Induction, and Detection of the HeLa Cellular Response to Anisomycin Treatment


®

Doc# 988-08338

Odyssey

®



Contents

Page


II. Apoptosis Assay Example ................................................................................2





®

-20• Anisomycin (Sigma

®

, Cat. # A9789)• Trypsin-EDTA Solution (1X) (Sigma

®

, Cat. # T-3924) • 37% formaldehyde• 10% Triton

®

X-100• Nunc™-Nalgene 96 Microwell™ Plate (VWR, Cat. # 167008) or Nunc™-Optical Bottom Plates 96-well

Black-walled/clear bottom with lid (VWR Cat. # 37000-558)• Secondary antibodies:

IRDye™ 800CW Goat anti-mouse (LI-COR, Cat. # 926-32210)*IRDye™ 800CW Goat anti-rabbit (LI-COR, Cat. # 926-32211)*IRDye™ 680 Goat anti-mouse IgG (H+L) (LI-COR, Cat. # 926-32220)*IRDye™ 680 Goat anti-rabbit IgG (H+L) (LI-COR, Cat. # 926-32221)*

• Odyssey

®

Blocking Buffer (LI-COR; Cat. #927-40000) or StartingBlock (PBS) blocking buffer (Pierce; Cat. # 37538).

Note:

When using primary antibodies discussed in this protocol, either blocking buffer will provide low background. This may not hold true for other primary antibodies.

• Primary antibodies:Cleaved Caspase-3 (Cell Signaling Technologies, Cat. # 9661)

β

-Tubulin (D-10), [Santa Cruz, Cat. # SC-5274] or Anti-

β

-Tubulin, clone AA2, [Upstate Group Inc., Cat. # 05-661]


®


LI-COR Biosciences


II. Apoptosis Assay Example

1. Allow HeLa (ATCC; CCL-2) cell growth in a T75 flask using standard tissue culture procedures until cells reach near confluency (~1.5x10

7


®

).

2. Remove growth media, wash cells with sterile 1X PBS, and displace cells with 5 mL Trypsin-EDTA (Sigma

®

).



5. Reconstitute cells in complete media such that 50,000 cells/ml is achieved.



8. Incubate cells and monitor cell density until ~ 80% confluency is achieved.


10. Add either serum free media for resting cells (mock) or serum-free media containing dilution series (1:2) of Anisomycin ranging in concentration from 0.07-40 µM. Add 100 µl of resting or activation media per well.

11. Transfer media from the dilution plate into the experimental plate.

12. Allow incubation at 37 °C for 4 hours.

13. When incubation period is complete, remove activation media manually or by aspiration. Immediately fix cells with

Fixing Solution

(4% formaldehyde in 1X PBS) for 20 minutes at room temperature (RT).

a. Prepare fresh

Fixing Solution

as follows:




Fixing Solution

(RT).

Add the

Fixing Solution



Odyssey

®



14. To permeabilize cells, wash five times with 1X PBS containing 0.1% Triton X-100 for 5 minutes per wash.

a. Prepare


as follows:


1X PBS + 0.1% Triton X-100 500 ml

b. Remove

Fixing Solution




(RT).


d. Allow plate to shake on a rotator for 5 minutes at RT.

e. Repeat washing steps 4 more times, removing wash manually each time.


15. Using a multi-channel pipettor, block cells/wells by adding 150 µl of LI-COR Odyssey Blocking Buffer or Pierce StartingBlock blocking buffer to each well.


Notes:





16. Allow blocking for 1.5 hours at RT with moderate shaking on a rotary shaker.

LI-COR Biosciences


17. Dilute the two primary antibodies in Odyssey or StartingBlock blocking buffer. Combine the following antibodies for Cleaved Caspase-3 target analysis:

• Cleaved Caspase-3; rabbit; 1:100 dilution; Cell Signaling Technology #9661

•

Normalizing antibody:

Anti-

β

-Tubulin, clone AA2; mouse; 1:100 dilution; Upstate #05-661 or

β

-Tubulin (D-10); mouse; 1:100 dilution; Santa Cruz Biotechnology #SC-5274


b. Remove blocking buffer and add 50 µl of the desired primary antibody or antibodies in Odyssey or StartingBlock blocking buffer to cover the bottom of each well.

c.


. To control wells, add 50 µl of Odyssey or StartingBlock blocking buffer only.


Notes:



a. Prepare


as follows:





(RT).


c. Allow plate to shake on a rotator for 5 minutes at RT.


20. Dilute the fluorescently labeled secondary antibody in Odyssey or StartingBlock blocking buffer and add 0.5% Tween-20 to the diluted antibody to lower background as specified below.

IRDye™ 680 goat anti-rabbit (1:200 dilution; LI-COR)IRDye™ 800CW goat anti-mouse (1:800 dilution; LI-COR)




Odyssey

®











• If you use plates other than those recommended above, the focus offset can be determined by scanning a plate containing experimental and control samples at 0.5, 1.0, 2.0, 3.0, and 4.0 mm focus offsets. Use the





b. Allow plate to shake on a rotator for 5 minutes at RT.

c. Repeat washing steps 4 more times, removing wash manually each time.


23. After final wash, remove wash solution completely from wells. Turn the plate upside down and tap or blot gently on paper towels to remove traces of wash buffer. For best results, scan plate immediately; plates may also be stored at 4 °C for several weeks (protected from light).


25. Scan the plate with detection in both the 700 and 800 channels using the Odyssey Infrared Imaging System (700 nm detection for IRDye™ 680 antibody and 800 nm detection for IRDye™ 800CW antibody). Use medium scan quality, 169 µm resolution, 3.0 mm focus offset, and an intensity setting of 5 for both 700 and 800 nm channels.

LI-COR Biosciences


same intensity settings for each scan. After reviewing the collected scans, use the focus offset with the highest signal-to-noise as your focus offset for experiments.






β-Tubulin normalizing antibody (IRDye™ 800CW)

0.07 0.15 0.30 0.60 1.25 2.50 5.0 10.0

Anisomycin, µM

Induction of apoptosis in HeLa cells was achieved with increasing concentrations of anisomycin. An increase in Cleaved Caspase-3, a cleaved by-product indicative of apoptosis, is illustrated in the graph. The ultimate result of apoptosis induction is cell death. This can clearly be seen at high concentrations of aniso-mycin (5-40 µM) in this example. The reduc-tion in cell number per well is taken into account when normalizing with another anti-body or a DNA stain. In an assay such as this normalization is very important.

20.0

duplicate rows

40.0Bac

kgro

un

d

1° &

2°

on

ly

Cleaved Caspase-3 detection antibody(Alexa Fluor 680)

Microplate Row

Row 1

Row 2

Anisomycin Induction of HeLa Cells

Anisomycin, µM

800

600

400

200

00.07 0.3 1.25 5 20

Inte

gra

ted

Inte

nsi

ty

4308




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LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, inc. Tween is a registered trademark of ICI Americas, Inc. Triton is a registered trademark of Union Carbide Chemicals and Plastics Corp. Sigma is a registered trademark or registered trademarks of Sigma-Aldrich Inc. Nunc and Microwell are trademarks of Nunc A/S Corporation. Falcon is a trademark of Becton Dickinson and Company. Gibco and Alexa Fluor are registered trademarks of Invitrogen Corporation. The Odyssey Infra-red Imaging System is covered by U.S. patent (6,495,812), foreign equivalents, and patents pending.

®

Odyssey


®


Phospho-p53 Detection in COS Cells in Response to Hydroxyurea

Published January, 2006. The most recentversion of this protocol is posted at http://biosupport.licor.com/support

®

Doc# 988-08339

Odyssey

®



Contents

Page


II. Assay Example .................................................................................................1




Odyssey

®

Reagents


®

Blocking Buffer (927-40000)

Additional Reagents

• 1X PBS wash buffer• Tissue culture reagents (serum, D-MEM, trypsin, 1X PBS)• Clear or black 96-well microplate (see


)• Hydroxyurea (Sigma

®

, Cat. # H8672)• Anti-phospho-p53 (Cell Signaling Technology, Cat. # 9286)• Normalization antibody: Anti-total ERK1 (Santa Cruz Biotechnology, Cat. # SC-94)• 20% Tween

®


®

X-100


®


II. Assay Example

1. Allow Cos-7 (ATCC; CRL-1651) cell growth in a T75 flask using standard tissue culture procedures until cells reach near confluency (~1.5x10

7


®

).





LI-COR Biosciences





9. Incubate cells and monitor cell density until 80 - 90% confluency is achieved (approximately 72 hours).

10. Warm serum-free media (D-MEM; Gibco) to 37°C.


12. Add either serum free media for resting cells (mock) or serum free media with serial concentrations of Hydroxyurea ranging 0.04 - 20 mM for activated cells. Add 100 µl of resting/activation media per well.

13. Allow incubation at 37°C for 7.5 minutes.

14. Remove activation or stimulation media manually or by aspiration. Immediately fix cells with

Fixing Solution

(4% formaldehyde in 1X PBS) for 20 minutes at room temperature.

a. Prepare fresh

Fixing Solution

as follows:




Fixing Solution


Add the

Fixing Solution



15. To permeabilize, wash five times with 1X PBS containing 0.1% Triton X-100 for 5 minutes per wash.

a. Prepare


as follows:


1X PBS + 0.1% Triton X-100 500 ml

b. Remove

Fixing Solution




(RT).


d. Allow plate to shake on a rotator for 5 minutes at RT.

e. Repeat washing steps 4 more times, removing wash manually each time.


Odyssey

®





Notes:





17. Allow blocking for 1.5 hours at RT with moderate shaking on a rotator.

18. Dilute the two primary antibodies in Odyssey Blocking Buffer. Combine the following antibodies for phospho-p53 target analysis, using total ERK1 for normalization:

• Phospho-p53 (Mouse; 1:400 dilution; Cell Signaling Technology Cat. # 9286) • Total ERK1 (Rabbit; 1:100 dilution; Santa Cruz Biotechnology Cat. # SC-94)



c.



19. Incubate with primary antibody overnight with gentle shaking at 4°C.


a. Prepare


as follows:





(RT).




LI-COR Biosciences


21. Dilute the fluorescently labeled secondary antibody in Odyssey Blocking Buffer as specified below. To lower background, add Tween-20 at a final concentration of 0.2% to the diluted antibody.

IRDye™ 800CW goat anti-rabbit (1:800 dilution; LI-COR Cat.# 926-32211)IRDye™ 680 goat anti-mouse (1:200 dilution; LI-COR Cat.# 926-32220)









c. Repeat washing steps 4 more times.


24. After final wash, remove wash solution completely from wells. Turn the plate upside down and tap or blot gently on paper towels to remove traces of wash buffer. For best results, scan plate immediately; plates may also be stored at 4 °C for several weeks (protected from light).


26. Scan the plate with detection in both the 700 and 800 channels using the Odyssey instrument (700 nm detection forIRDye™ 680 antibody and 800 nm detection for IRDye™ 800CW antibody). Use medium scan quality, 169 µm resolution, 3.0 mm focus offset, and an intensity setting of 5 for both 700 and 800 nm channels.

Odyssey

®











• If you use plates other than those recommended above, the focus offset can be determined by scanning a plate containing experimental and control samples at 0.5, 1.0, 2.0, 3.0, and 4.0 mm focus offsets. Use the same intensity settings for each scan. After reviewing the collected scans, use the focus offset with the highest signal-to-noise as your focus offset for experiments.


• Intensity for both 700 and 800 nm channels should be set to 5 for initial scanning. If your image signal is sat-urated or too high, re-scan using a lower intensity setting (i.e., 2). If your image signal is too low, re-scan using a higher intensity setting (i.e., 7).



LI-COR Biosciences




Two-color In-Cell Western detection of phospho-p53 induction in Cos-7 cells.

700 nm channel display of phospho-p53.

800 nm channel display of total ERK1 normalization.

0.04 0.08 0.16 0.31 0.63 1.25 2.5 5 20Hydroxyurea, mM

Background

Figure 1. Dose response of Cos-7 cells to Hydroxyurea as measured by specific antibody detecting phosphory-lated-p53 (Ser16) using total ERK1 for normalization. The image represents a 96-well two-color In-Cell Western with the 700 and 800 channels detecting phosphorylated-p53 (Ser16) and total ERK1, respectively. Background wells were incubated with secondary antibody but no primary antibody. The graph represents the average of four sets of quantitative data, demonstrating the percent induction of phosphorylated-p53 (Ser16).

10

duplicate rows

0.0

% in

du

ctio

n o

f p

53-

ph

osp

ho

ryla

tio

n (

ser1

6)

4308




www.licor.com

LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, inc. Tween is a registered trademark of ICI Americas, Inc. Triton is a registered trademark of Union Carbide Chemicals and Plastics Corp. Sigma is a registered trademark or registered trademarks of Sigma-Aldrich Inc. Nunc and Microwell are trademarks of Nunc A/S Corporation. Falcon is a trademark of Becton Dickinson and Company. Gibco and Alexa Fluor are registered trademarks of Invitrogen Corporation. The Odyssey Infra-red Imaging System is covered by U.S. patent (6,495,812), foreign equivalents, and patents pending.

®

Odyssey


®


Phospho-p38 Detection in HeLa Cells in Response to Anisomycin

Published September, 2007. The most recent version of this protocol is posted at http://biosupport.licor.com/support

®

Doc# 988-09368

Odyssey

®



Contents

Page


II. Assay Example .................................................................................................1




• IRDye

®

800CW- and IRDye

®

680- labeled secondary antibodies (LI-COR Biosciences)• Odyssey

®

Blocking Buffer (LI-COR Biosciences, Cat.# 927-40000)• 1X PBS wash buffer (LI-COR Biosciences, Cat.# 928-40018 or 928-40020)• Standard tissue culture reagents (serum, D-MEM media, Trypsin-EDTA, etc.)• HeLa cells (ATCC, Cat.# CCL-2)• Nunc™ 96 Microwell™ Plate (Nunc, Cat.# 167008)• Anisomycin (Sigma-Aldrich Cat.# A9789)• Anti-phospho p38 antibody (Cell Signaling Technology, Cat.# 9211)• Normalization antibody (e.g.: anti-total ERK2 (Santa Cruz Biotechnology, Cat.# sc-1647)• 20% Tween

®


®

X-100

II. Assay Example

1. Allow HeLa cell growth in a T75 flask, using standard tissue culture procedures, until cells reach near confluency (1.5x10

7

cells).




5. Resuspend cells in 20 mL of complete media and count cells using a hemacytometer.

6. Dilute cells with complete media such that 75,000 cells/mL is achieved.


8. Under sterile conditions, dispense 200 µL of the cell suspension per well in a Nunc™ 96 Microwell™ Plate (15,000 cells plated per well).

LI-COR Biosciences


9. Incubate cells at 37 °C with 5% CO

2

in air atmosphere; monitor cell density until confluency is achieved with well-to-well consistency (approximately two to three days).

10. Warm serum-free media (D-MEM; Gibco

®

) to 37 °C. In a fresh 96-well microplate, prepare two-fold serial dilutions of anisomycin, ranging from 2 to 1000 nM (0.5 to 265 ng/mL). Leave the first and second wells without anisomycin (resting cells as control), as shown in section


.


12. Transfer media and anisomycin dilutions from the dilution plate into the experimental plate.

13. Incubate plate at 37 °C for 30 minutes.

14. Remove stimulation media manually or by aspiration. Immediately fix cells with 3.7% formaldehyde for 20 minutes at room temperature.

a. Prepare fresh

Fixing Solution

as follows:

1X PBS 45 mL37% Formaldehyde 5 mL

3.7% Formaldehyde 50 mL

b. Using a multi-channel pipettor, add 150 µL of fresh

Fixing Solution


Add the

Fixing Solution




a. Prepare


as follows:

1X PBS 495 mL10% Triton X-100 5 mL

1X PBS + 0.1% Triton X-100 500 mL

b. Remove

Fixing Solution


c. Using a multi-channel pipettor, add 200 µL of


(RT).





Odyssey

®



16. Using a multi-channel pipettor, block cells/wells by adding 150 µL of LI-COR Odyssey Blocking Buffer to each well.


Notes:

• No single blocking reagent will be optimal for every antigen-antibody pair. Some primary antibodies may exhibit greatly reduced signal or different nonspecific banding in different blocking solutions. If you have difficulty detecting your target protein, changing the blocking solution may dramatically improve perfor-mance. If the primary antibody has worked well in the past using chemiluminescent detection, try that blocking solution.





18. Add the two primary antibodies to a tube containing Odyssey Blocking Buffer. Combine the following solutions as defined below for phospho-p38 target analysis, using total ERK2 for normalization:

• Phospho-p38 (rabbit; 1:100 dilution; Cell Signaling Technology Cat. # 9211)

• Total ERK2 (mouse; 1:100 dilution; Santa Cruz Biotechnology Cat. # sc-1647)


b. Remove blocking buffer from the blocking step and add 50 µL of the desired primary antibody or antibodies in Odyssey Blocking Buffer to cover the bottom of each well.

c.


. Add 50 µL of Odyssey Blocking Buffer only to control wells.


Note:


LI-COR Biosciences



a. Prepare


as follows:

1X PBS 995 mL20% Tween-20 5 mL

1X PBS with 0.1% Tween-20 1000 mL

b. Using a multi-channel pipettor add 200 µL of


(RT).





Goat anti-rabbit; IRDye 800CW (1:800 dilution; LI-COR)Goat anti-mouse; IRDye 680 (1:200 dilution; LI-COR)



22. Mix the antibody solutions well and add 50 µL of the secondary antibody solution to each well. Incubate for 60 minutes with gentle shaking at RT. Protect plate from light during incubation.


a. Using a multi-channel pipettor, add 200 µL of







25. Before plate scanning, clean the bottom plate surface with moist lint free paper to avoid any obstructions during scanning.

26. Scan the plate with detection in both the 700 and 800 channels using the Odyssey Infrared Imaging System (700 nm detection for IRDye 680 antibody and 800 nm detection for IRDye 800CW antibody). Use medium quality, 169 µm resolution, 3.0 mm focus offset, and an intensity setting of 5 for both 700 and 800 nm channels (optimization may be required.

Odyssey

®








96 well, clear Nunc™ (Part Number 167008, 161093)96 well, clear Falcon™ (Part Number 353075, 353948)96 well, black with clear bottom Nunc™ (Part Number 165305)

384 well, clear Nunc™ (Part Number 164688, 164730)384 well, clear Falcon™ (Part Number 353961, 353962)384 well, black with clear bottom Nunc™ (Part Number 142761)


• If you use plates other than those recommended above, the focus offset can be determined by scanning a plate containing experimental and control samples at 0.5, 1.0, 2.0, 3.0, and 4.0 mm focus offsets. Use the same intensity settings for each scan. After reviewing the scans, use the focus offset with the highest signal-to-noise for your experiments.




LI-COR Biosciences




Two-color In-Cell Western detection of phospho-p38 induction in HeLa cells.

800 nm image display of phospho-p38.

700 nm channel display of total ERK2 normalization.

2 4 8 16 31 63 125 250 1000Anisomycin, nM

Background

Figure 1. Dose response of HeLa cells to Anisomycin as measured by specific antibody detection of phos-phorylated-p38 (Thr180/Tyr182), using total ERK2 for normalization. The image represents a 96-well two-color In-Cell Western with the 800 and 700 channels detecting phosphorylated-p38 (Thr180/Tyr182) and total ERK2, respectively. Background wells were incubated with secondary antibody but no primary antibody. The graph represents the average of eight sets of quantitative data, demonstrating the percent induction of phosphorylated-p38 (Thr180/Tyr182).

500

duplicate rows

0.0

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LI-COR is an ISO 9001 registered company. © 2007 LI-COR Inc. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, inc. Gibco is a registered trademarks of Invitrogen Corporation. Tween is a registered trademark of ICI Americas, Inc. Triton is a registered trademark of Union Carbide Chemicals and Plastics Corp. Nunc and Microwell are trademarks of Nunc A/S Corporation. Falcon is a trademark of Becton Dickinson and Company. The Odyssey Infrared Imaging System is covered by U.S. patents, foreign equivalents, and patents pending.

®

Odyssey


®


Complete Sample Protocol for PMA-induced ERK Activation in Suspension Cell Lines


®

Doc# 988-08340

Odyssey

®



Contents

Page


II. Experimental Design ........................................................................................2

III. Assay Example .................................................................................................2

IV. Experimental Considerations............................................................................6

V. Experimental Results........................................................................................7


• 1X PBS wash buffer• Tissue culture reagents (RPMI 1640, fetal bovine serum, etc.)

• Round-bottom 96-well plates: BD Bioscience (cat# 353077)• Jurkat cells: ATCC

®

(Cat.# TIB-152™)• THP-1 monocytes: ATCC (Cat.# TIB-202™)• K-562 lymphocytes: ATCC (Cat.# CCL-243™)• Concentrated Prefer (5x): Anatech LTD (Cat.# 411)• Odyssey

®

Blocking Buffer: LI-COR Biosciences (Cat.# 927-40000)• TO-PRO

®

-3: Molecular Probes (Cat.# T-3605)• PMA (phorbol 12-myristate 13-acetate): Sigma

®

(Cat.# P1585)• DMSO (dimethyl sulfoxide): Sigma

®

(Cat.# D8418)• ERK rabbit antibody: Santa Cruz (Cat.# sc-94)• pERK mouse antibody: Cell Signaling Technology (Cat.# 9106)• IRDye™ 680-labeled goat anti-mouse secondary antibody: LI-COR (Cat.# 926-32220)*• IRDye™ 800CW-labeled goat anti-rabbit secondary antibody: LI-COR (Cat.# 926-32211)* • Large Western Incubation Box: LI-COR (Cat.# 929-97803)


®


LI-COR Biosciences


II. Experiment Design

III. Assay Example

1. Allow Jurkat (ATCC; TIB-152) cells to grow in a T75 flask using standard tissue culture procedures. Avoid growing cells to density greater than 2x10

6

cells.

2. Transfer cells in growth media to 50 ml conical tubes and centrifuge at 500xg for 5 minutes.

3. Remove media and resuspend cell pellet in 10 ml of serum free media (pre-warmed to 37 °C). Pipet very slowly in order to maintain cell integrity while disrupting the cell pellet. Transfer resuspended cells into T75 flask and place in an incubator (37 °C and 5% CO

2

).

4. Allow cells to settle down for 30 minutes before taking a 50 µl aliquot of cells for cell counting using a hemacytometer.

654321 121110987

A

B

C

D

E

F

G

H

0.0051°Ab2°Ab

0.0051°Ab2°Ab

0.0051°Ab2°Ab2°Ab

DMSOBkgd

2°AbDMSOBkgd

2°AbDMSOBkgd

0.005

1°Ab

2°Ab

PMA (ng/mL)Primary AntibodySecondary Antibody

1°Ab2°Ab

DMSO

1°Ab2°Ab

DMSO

1°Ab2°Ab

DMSO

0.0131°Ab2°Ab

0.0131°Ab2°Ab

0.0131°Ab2°Ab

0.0251°Ab2°Ab

0.051°Ab2°Ab

0.11°Ab2°Ab

0.21°Ab2°Ab

0.41°Ab2°Ab

0.81°Ab2°Ab

1.61°Ab2°Ab

3.21°Ab2°Ab

0.0251°Ab2°Ab

0.0251°Ab2°Ab

0.051°Ab2°Ab

0.051°Ab2°Ab

0.11°Ab2°Ab

0.11°Ab2°Ab

0.21°Ab2°Ab

0.21°Ab2°Ab

0.41°Ab2°Ab

0.41°Ab2°Ab

0.81°Ab2°Ab

0.81°Ab2°Ab

1.61°Ab2°Ab

1.61°Ab2°Ab

3.21°Ab2°Ab

3.21°Ab2°Ab

Figure 1. Experimental design for Figure 2. Round bottom 96-well plate: ~200,000 Jurkat cells per well (C1-E12) in triplicate samples.

Important: It is the serum withdrawal from the complete media that allows suspension cells to attach to plates (i.e., T75 flask). Gravity will cause cells to form a monolayer over time (10 to 15 minutes). Once a monolayer is formed, the rest of cells in the serum-free media will remain in suspension and will not further attach to the plates once a monolayer of cells are established. Only the cells in suspension in the T75 flask will be used in the following steps.

Odyssey

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5. Add appropriate volume of serum free media such that 1x10

6

cells/ml is achieved (1 plate x 96 wells x 200 µl of cells/well = ~20ml/plate).

6. Serum deprive cells by replacing cells suspended in serum free media back in the incubator for an additional 3.5 hours or overnight.

7. Add 2 µl of DMSO for both the background samples (serves as non-specific background fluorescence) and resting cells (serves as basal control) in triplicate wells. Add 2 µl of 1:1 serial dilutions of PMA ranging from 0.005 to 3.2 ng/ml in triplicate wells.

8. Using a multi-channel pipettor, transfer 200 µl of suspended cells (~200,000 cells) per well into the wells containing 2 µl of DMSO or PMA from step 7.

9. Allow incubation at 37 °C for 15 minutes.

10. Directly add to the cell suspension, 50 µl of concentrated (5x) Prefer (or 25 µl of 37% formaldehyde; final concentration is 4%) into each well.

11. Allow cells to fix for 20 minutes at room temperature with very gentle rotation (set at speed 2 on The Belly Dancer; Stovall).

12. Centrifuge at 1500 rpm (332 rcf) for 10 minutes.

Important note about choosing round bottom plates for suspension cell assays: If imaging with Aerius, LI-COR recommends clear, round bottom 96-well plates from BD Bioscience (Cat.# 353077) or from Nunc (Cat.# 167008). If imaging with Odyssey, BD Bioscience plates (Cat.# 353077) should be used. Imaging Nunc round bottom plates with Odyssey will yield quantitatively accurate results, but the images will not be as visually satisfying and over all signal intensity will be lowered. This is due to differences in the optical properties of the round bottom wells.

Important: Be careful not to disrupt the cells during this PMA-induced activation step. During this critical step, cells will sediment to the bottom of the wells by gravity forming a monolayer. This monolayer can be easily viewed under a light source. The monolayer will appear opaque rather than transparent. Clumping of cells will lead to detachment from plates during incubation and washing steps. Be careful of handling the plate at this stage because the cells will be very loosely attached to the bottom of the wells.

Important: Gently add Prefer into wells using side of the wells to avoid detaching the cells from the well bottom. During fixation, the cell monolayer will attach more firmly to the wells. However, the strength of the attachment is never as strong as adherent cells grown on plates. Thus, a degree of caution is needed during every step of this procedure.

LI-COR Biosciences


13. To permeabilize cells, wash three times with 100 µl of 1x PBS containing 0.1% Triton X-100 for 5 minutes each by centrifugation at 1500 rpm (332 rcf).

a. Prepare

Triton Permeabilization Solution

as follows:


1x PBS + 0.1% Triton X-100 500 ml

b. Remove fixing solution (if using formaldehyde, collect in an appropriate waste container).

c. Using a multi-channel pipettor, add 100 µl of fresh


.

Add the solution carefully by pipetting down the sides of the wells to avoid detaching the cells from the well bottom.

d. Centrifuge at 1500 rpm (332 rcf) for 5 minutes.

e. Gently remove


by manually pipetting.

f. Repeat washing step two more times.


Note:

If detecting cell surface proteins with exofacial antigens, you do not need to permeabilize cells. After fixing cells in step 11, wash cells (see step 18) before proceeding to blocking (step 14).

14. Using a multi-channel pipettor, block cells/wells by adding 100 µl of Odyssey Blocking Buffer to each well.

Add the solution carefully by pipetting down the sides of the wells to avoid detaching the cells

.

15. Allow blocking for 1 hour at room temperature with very gentle shaking on a rotator.

16. Dilute the two primary antibodies in Odyssey Blocking Buffer. Combine the following antibodies for ERK target analysis:

• Rabbit anti-ERK antibody (1:200 dilution; Santa Cruz) • Mouse anti-phospho-ERK antibody (1:100 dilution; Cell Signaling Technology)


b. Remove blocking buffer from the blocking step.

c. Add 50 µl of Odyssey Blocking Buffer only to the background control wells (serves as non-specific background fluorescence).

d. Add 50 µl of the primary antibody solution into rest of wells.

17. Incubate with primary antibody for 2 hours at room temperature or overnight at 4 °C with very gentle shaking on a rotator.

Odyssey

®



18. Wash the plates five times with 200 µl of 1x PBS + 0.1% Tween-20 for 5 minutes by centrifugation at 1500 rpm (332 rcf).

a. Prepare


as follows:



b. Remove primary antibody solution.



.


d. Centrifuge at 1500 rpm (332 rcf) for 5 minutes.

e. Gently remove



f. Repeat washing steps 4 more times.

19. Dilute the fluorescently labeled secondary antibody in Odyssey Blocking Buffer with 0.2% Tween-20 in order to lower background as specified below.

IRDye™ 680 goat anti-mouse (1:200 dilution; LI-COR)IRDye™ 800CW goat anti-rabbit (1:800 dilution; LI-COR)



20. Mix the antibody solutions well and add 50 µl of the secondary antibody solution to each well. Incubate for one hour with very gentle shaking on a rotator at room temperature.

Protect plate from light during incubation.

Use a large black Western Incubation Box (LI-COR) to protect plate from light during subsequent steps.

21. Wash the plates five times with 200 µl of 1x PBS + 0.1% Tween-20 at room temperature for 5 minutes by centrifugation at 1500 rpm (332 rcf).

a. Remove secondary antibody solution.



. Make sure to carefully add the solution down the sides of the wells to avoid detaching the cells.

c. Centrifuge at 1500 rpm (332 rcf) for 5 minutes.

d. Gently remove



e. Repeat washing step four more times.


22. After final wash, remove wash solution completely from wells. For best results, scan plate immediately; plates may also be stored at 4 °C for several weeks (protected from light).

LI-COR Biosciences


IV. Experimental Considerations


• Clear round bottom plates are recommended for In-Cell Western analysis of non-adherent cells:

Odyssey Infrared Imaging System

96 well format BD Bioscience (Cat.# 353077)

Note:

Clear round bottom plates from Nunc (cat# 163320) can also be used and will yield quantitatively accurate data. However, the images may be less visually satisfying when imaged on Odyssey due to differences in the optical properties of the round bottom wells. The Nunc plates work well on the Aerius system.

Aerius Automated Infrared Imaging System

96 well format BD Bioscience (Cat.# 353077)Nunc (Cat.# 167008)

• Both the flat and round bottom plates show some plate autofluorescence. However, the plate autofluo-rescence is relatively small compared to the actual signal.When using the recommended BD Bioscience round bottom plate, the recommended focus offset is 3.0 to 3.5 mm. The recommended focus offset for the Nunc round bottom plate is 3.5 to 3.95.

• If you use plates other than those recommended above, the focus offset can be determined by scanning a plate containing experimental and control samples at 0.5, 1.0, 2.0, 3.0, and 4.0 mm focus offsets. Use the same intensity settings for each scan. After reviewing the collected scans, use the focus offset with the highest signal-to-noise as the focus offset for experiments, or fine-tune the focus offset in smaller increments if desired.


• Intensity for both 700 and 800 nm channels should be set to 5 for initial scanning. If your image signal is saturated or too high, re-scan using a lower intensity setting (i.e., 2.5). If your image signal is too low, re-scan using a higher intensity setting (i.e., 7.5).

• 200 µm (Aerius) or 169 µm (Odyssey) scanning resolution provides satisfactory results with minimal scan time. Higher resolution may be used, but scan time will increase.

• Establish the specificity of your primary antibody by screening lysates through Western blotting. If signif-icant non-specific banding is present, choose alternative primary antibodies. Non-specific binding of primaries will complicate interpretation of In-Cell Western assay results.

23. Place the plate on the Odyssey scan surface and scan with detection in both the 700 and 800 channels (700 nm detection for IRDye™ 680 antibody and 800 nm detection for IRDye™ 800CW antibody). Use 169 µm resolution, 3.0 to 3.5 mm focus offset for BD Bioscience plate (3.5 to 3.95 for Nunc plate), and an intensity setting of 5 or less for both 700 nm and 800 nm channels.

Odyssey

®



V. Experimental Results


Two-color In-Cell Western detection of phospho-ERK induction in Jurkat cells.

700 nm channel display of phospho-ERK.

800 nm channel display of total ERK normalization.

0.005 0.013 0.025 0.05 0.1 0.2 0.4 0.8 3.2

phorbol 12-myristate 13-acetate (ng/ml)Background

Figure 2. Dose response of Jurkat cells to phorbol 12-myristate 13-acetate (PMA) as measured by specific antibody detecting dual-phosphorylated ERK (Thr202/Tyr204). The image represents a 96-well two-color In-Cell Western with the 700 and 800 channels detecting phosphorylated and total ERK, respectively. The image was scanned using the Aerius imaging system (similar to Odyssey) with scan setting of 200 µm resolution, focus offset of 3.5, and intensity of 2 (700 channel) and 4 (800 channel). Background wells were incubated with secondary antibodies but no primary antibodies. The graph represents normalized quan-titative data demonstrating the increase in ERK phosphorylation in response to PMA stimulation.

1.60.0

LI-COR Biosciences


Two-color In-Cell Western detection of phospho-ERK induction.

700 nm channel display of phospho-ERK.

800 nm channel display of total ERK normalization.

0.1 3.2 ng/ml PMA

Jurkat

Figure 3. ERK activation in Jurkat, K-562 and THP-1 non-adherent cells in response to PMA as measured by specific antibody detecting dual-phosphorylated ERK (Thr202/Tyr204). The image represents a 96-well two-color In-Cell Western with the 700 and 800 channels detecting phosphorylated and total ERK, respectively. The image was scanned using the Aerius imaging system (similar to Odyssey) with scan setting of 200 µm resolution, focus offset of 3.5, and intensity of 3.5 (700 channel) and 5 (800 channel). Background (B) wells were incubated with secondary antibodies but no primary antibodies. The graph represents nor-malized quantitative data demonstrating the increase in ERK phosphorylation in response to PMA stimulation.

0.0B 0.1 3.2

K-5620.0B 0.1 3.2

THP-10.0B

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LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, inc. Alexa Fluor and TO-PRO are registered trademarks of Invitrogen. ATCC, TIB-152, TIB-202, and CCL-243 are trademarks or registered trademarks of American Type Culture Collection. Tween is a registered trademark of ICI Americas, Inc. Sigma is a registered trademark or registered trademarks of Sigma-Aldrich Inc. The Odyssey Infrared Imaging System is covered by U.S. patent (6,495,812), foreign equivalents, and patents pending.

®

Odyssey


®

Technical Note

FAQs for Suspension Cells for ICW Protocols


®

Doc# 988-08341

FAQs For Suspension Cells For In-Cell Western Protocols


1. Handling Suspension Cells

1.1 How do you culture suspension cells?

For instructions on culturing suspension cells, look up your cell line at http://www.atcc.org/ and follow the guidelines.

1.2 How do you make non-adherent cells (suspension cells) attach to plates?

A simple trick is to replace your complete media containing 10% serum (usually fetal bovine serum) with the same media minus the serum. Then allow the cells to sediment, forming a mono-layer of cells within 10 minutes.

Caution:

although cells appear attached to the plates, they are relatively loosely attached and therefore, extreme caution is required during solution changing steps.

1.3 How do I know that I have a monolayer?

Method #1

– Examine cells in the round bottom 96-well plates under a light microscope. The center of the wells should all have a small flat circular surface area where all the cells in that field are “in focus”. Moving the plane of focus, up or down, will cause cells to be “off focus”.

Method #2

– Hold the round bottom 96-well plate under a light source. The monolayer should look opaque rather than transparent. Cells will not attach on top of the cell monolayer, so the opaqueness is due only to the monolayer.

1.4 I cannot get a monolayer of cells. I get lots of spaces between cells. Is seeding 200,000 cells/well enough?

Seeding 200,000 cells/well is more than enough to form a complete cell monolayer. It is neces-sary to allow the cells in serum-free media to sediment in the T75 flask (or other tissue culture plates) for approximately 30 minutes before counting cells using a hemacytometer. When cells in serum-free media are placed, for example, in a T7 tissue culture flask, a monolayer of cells will immediately begin to form on the bottom of the flask. This will dramatically decrease the number of cells in suspension that are available for plating. Note: Once a complete monolayer has formed on the plate, the rest of cells will remain in suspension. Count these cells in suspension and the cells attached to the T75 flask can be discarded later.

1.5 During my washing steps, cells are coming off the plates.

1.5.1 Are you using the recommended round bottom 96-well plate [BD Bioscinece; Part Number 353077]?

If no

, cells will more easily detach from the flat bottom plates than the round bottom plates. The multi-channel pipettors will generate enough pressure when expelling liquid from the pipet to cause cell detachment when using flat bottom plates. Cells will detach even when pipetting down the sides of the wells.

If yes

, make sure you pipet down the sides of the wells and not directly onto the cells. If this doesn’t help, you may need to change your multi-channel pipettor because different brands of pipettors have different amount of pressure required to expel the liquid from the pipet. The recommended multi-channel pipettor is the 12-channel Finnpipette [Thermo Electron Corp; Part Number 4610050].

LI-COR Biosciences


1.5.2 Are you shaking or rotating the plates at a moderate to high speed?

If yes

, gentler shaking/rotating is needed to prevent cells from detaching. Cells will detach. Set shaking or rotating speed to very low speed.

If no

, are you dumping the solutions straight from the plates? Dumping causes cells to detach. Either aspirate very slowly or manually pipet using the sides of the wells.

2. Round vs. Flat Bottom 96-well Plates

2.1 Why can’t I use the flat bottom 96-well plates?

LI-COR Biosciences recommends using the round bottom 96-well plates. For an explanation, see 1.5.

2.2 When I scan an empty round bottom 96-well plate, I get lots of background noise.

The round bottom plate shows some background autofluorescence. The background fluorescence is relatively small compared to signal (about 200-fold difference depending on the intensity of the signal) and can be subtracted from the signal. It is necessary to include background wells contain-ing cells and only the secondary antibodies in order to completely subtract away the background noise originating from the plate as well as from the non-specific binding of the secondary anti-bodies.

3. Scan Settings

3.1 Why does my canned image look so weak?

Assuming that you followed the protocol correctly and your antibodies work, did you set the focus offset to 3.0 to 3.5 mm for the BD Bioscience round bottom plates (Part Number 353077)? If using the Nunc round bottom plates (Part Number 16332), the default setting for the flat bottom 96-well plates (3.0 mm) will not produce much signal. The focus offset for the Nunc round bot-tom plates should be set to 3.5 to 3.95mm. For maximum signal strength with Odyssey, BD Bio-science round bottom plates are recommended. Both BD Bioscience and Nunc round bottom plates work well with Aerius.

4. Other Suspension Cell Lines and Different Pathways

4.1 Have you tested other suspension cell lines?

Yes. Suspension cell lines tested include Jurkat, K-562 and THP-1. A sample protocol can be downloaded from http://biosupport.licor.com/support.

4.2 Have you tested other pathways?

Yes. Pathways tested include ERK activation and apoptosis using cleaved caspase3 as a marker (Figure 1). A sample protocol can be downloaded from http://biosupport.licor.com/support.

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LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey is a trademark of LI-COR, inc. Nunc is a trademark of Nunc A/S Corporation. The Odyssey Infrared Imaging System is covered by U.S. patent (6,495,812), foreign equivalents, and patents pending.

Two-color In-Cell West-ern detection of cleaved caspase-3.

700 nm channel display of TO-PRO-3 for normal-ization.

800 nm channel display of cleaved caspase-3.

1 10 µM Anisomycin

Jurkat Cells

Figure 1. Anisomycin-induced apoptosis in Jurkat cells. The image represents a 96-well two-color In-Cell Western with the 700 and 800 channels detecting TO-PRO-3 DNA staining and cleaved caspase-3 (Asp175), respectively. The image was scanned using the Aerius imaging system with scan setting of 200 µm resolution, focus offset of 3.5, and intensity of 3.5 (700 channel) and 4 (800 chan-nel). Background (B) wells were incubated with a second-ary antibody but no primary antibody. The graph represents normalized quantitative data demonstrating the increase in caspase-3 cleavage in response to aniso-mycin treatment for 3 hours in Jurkat cells.

0B

®

Odyssey


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®

Electrophoretic Mobility Shift Assay (EMSA) Using IRDye Oligonucleotides

Doc# 982-07487

Revised May, 2004. The most recent version of this protocol, with color figures, is posted at http://biosupport.licor.com/support

™

EMSA Using IRDye™ Oligonucleotides


I. Introduction

Gel shift assays or electrophoretic mobility shift assays (EMSA) provide a simple method to study DNA-pro-tein interactions. This assay is based on the principle that a DNA-protein complex will have different mobility during electrophoreses than non-bound DNA. These shifts can be visualized on a native acryla-mide gel using labeled DNA to form the DNA-protein binding complex. To date, protocols require labeling DNA by radioisotope (1), digoxygenin (2), or biotin (3). The Odyssey

®

Infrared Imaging System (LI-COR

®

Biosciences) offers a quick and easily-adapted alternative method to radioisotopic and chemiluminescent detection methods for EMSA analysis and visualization.

A DNA oligonucleotide end-labeled with a LI-COR IRDye™ infrared dye is a good substrate for protein binding. Two IRDyes™ (IRDye™ 700 phosphoramidite and IRDye™ 800 phosphoramidite) are available for labeling DNA fragments, allowing the study of competitive binding of a protein to two DNA fragments simultaneously in the same reaction. IRDye™-labeled DNA detection is linear within a 50-fold dilution range, from 9.1 fmol to 0.18 fmol. Additional benefits include no hazardous radioisotope, no gel transfer to membrane or gel drying, no chemiluminescent substrate reagents, and no film exposure. Following electrophoresis, the gel can be imaged while remaining in the glass plates. If necessary, the gel can be placed back in the electrophoresis unit and run longer.

Existing mobility shift assay protocols can be easily transformed into infrared assays by replacing the exist-ing DNA oligonucleotides with IRDye™ end-labeled oligonucleotides. The binding conditions and elec-trophoresis conditions will remain the same as with any other EMSA detection method.

II. General Methodology

IRDye™ labeling of DNA fragments

To obtain IRDye™ end-labeled DNA fragments, IRDye™- labeled oligos are used. It is critical that the DNA fragment is end-labeled rather than having dye incorporated into the DNA, which interferes with the formation of the DNA-Protein complex.

Synthetic oligonucleotides 5’ end-labeled with IRDye™ 700 phosphoramidite or IRDye™ 800 phosphoramidite are available from LI-COR Biosciences (Express Primers, part numbers 4200-31 and 4200-31B). Oligonucleotides are manufactured in single strand form; therefore, both forward and reverse DNA oligonucleotides must be purchased.

Once oligonucleotides are obtained, they need to be annealed to form a double-stranded DNA fragment. Oligonucleotides are annealed by placing the oligonucleotide set in a 100°C heat block for 5 minutes, leaving the oligonucleotides in the heat block and turning it off to slowly cool to room temperature.

Important: Both oligonucleotide sequences should be end-labeled with the same IRDye™. There is a significant decline (~70%) in signal intensity when using only one end-labeled oligonucleotide.

LI-COR Biosciences


Binding Reaction

A universal binding condition that applies to every protein-DNA interaction cannot be recommended, since binding conditions are specific for each protein-DNA interaction. Thus, the user should establish binding reaction conditions for each protein-DNA pair. Binding buffer should be the same for this method as with any other mobility shift detection method used.

After the addition of DNA to the protein-buffer mix, reactions are incubated to allow protein to bind to DNA. Time required for binding is the same as when radioactively-labeled DNA fragments are used; a typical incubation condition is 20-30 minutes at room temperature. Since IRDyes™ are sensitive to light, it is best to keep binding reactions in darkness during incubation periods (e.g., put tubes into a drawer or simply cover the tube rack with aluminum foil). After the incubation period, native loading dye is added to the binding reaction.

NOTE:

In some cases, we observed that DNA control reactions (no protein) have lower signal than reac-tions containing protein. This may be due to lower stability of the dye in certain buffer conditions. The addition of 5 mM DTT and 0.5% Tween

®

to all reactions reduces this phenomenon.

Important: It is critical not to use any blue loading dye (e.g. bromophenol blue), as this will be visible on the Odyssey image. Use 10X Orange loading dye instead (LI-COR part number 927-10100).

Figure 1. AP-1 EMSA using IRDye 700 end-labeled oligonucleotide duplex.

It is common to use unlabeled DNA duplex to determine binding specificity. Excess unlabeled DNA is added to the binding reaction; therefore, it competes with the labeled DNA for binding sites. If competition eliminates labeled DNA binding, no shift is observed (see last three lanes in gel), indi-cating that the binding reaction is specific.

Competition reactions contained 100-fold molar excess of wild-type oligonucleotide duplex. Nuclear extracts of Hela, Hela 2 hour serum response, and Hela 4 hour serum response were used to visualize and increase in AP-1 binding as a result of the serum response treatment to the Hela cells.

No

Extr

act

Hel

aH

ela-

2hr S

RH

ela-

4 hr

SR

Hel

aH

ela-

2hr S

RH

ela-

4 hr

SR

Nuclear Extract - + + + + + +

AP-1 IRDye™ 700 oligo + + + + + + +

AP-1 wild-type competitor oligo - - - - + + +



.

Electrophoresis

Separation of protein-DNA complexes is usually performed by loading binding reactions onto native poly-acrylamide gels. Percentage of the gel depends on the protein size and DNA fragment, but a 5% gel is a good starting point. Based on the result obtained, use a lower (e.g. 4%) or higher percent gel. Electro-phoresis is usually performed at 10 V/cm at room temperature, or at 4°C in Tris-acetate, Tris-borate, or Tris-glycine-EDTA gel and buffer.

NOTE:

For best results, perform electrophoresis in the dark (simply put a cardboard box over the electro-phoresis apparatus).

Scanning

There is no need to remove the gel from the glass plates. This makes gel handling easier and allows running the gel further, if needed, after scanning is completed. Possible deformations or tearing of the gel while separating plates are also eliminated.

AP-1 consensus/mutant binding AP-1 consensus binding AP-1 mutant binding700 nm/800 nm image 700 nm image 800 nm image

Figure 2. AP-1 EMSA using 2.5 µg Hela 4-hour serum response nuclear extract to demonstrate binding specificity of AP-1 consensus DNA duplex. Binding specificity determination using Odyssey 2-color imaging. (A copy of this docu-ment with color figures can be downloaded from http://support.licor.com.)

Competition using mutant DNA duplexes is another common method to determine binding specificity. A mutant DNA sequence is used to compete with the wild-type binding sequence. Specific binding is observed when mutant DNA (unlabeled) does not reduce the binding of labeled wild-type DNA. Two-color analysis of mutant vs. wild-type binding is done using the Odyssey. The wild-type oligos are labeled with IRDye™ 700 phosphoramidite and mutant oligos with IRDye™ 800 phosphoramidite. In the figure above, the mutant non-specific binding is very intense (800 nm image); however, there is no decrease in wild-type binding (700 nm image).

Lane 1 – Free AP-1 consensus oligonucleotide IRDye™ 700 end-labeled and AP-1 mutant oligonucleotide IRDye™ 800 end-labeled with no nuclear extract; Lane 2 – Nuclear extract with 0:1 ratio of AP-1 consensus oligonucleotide IRDye™ 700 end-labeled to AP-1 mutant oli-gonucleotide IRDye™ 800 end-labeled; Lane 3 – Nuclear extract with 1:0 ratio of AP-1 consensus to mutant oligonucleotide; Lane 4 – Nuclear extract with 1:1 ratio of AP-1 consensus to mutant oligonucleotide; Lane 5 – Nuclear extract with 1:2 ratio of AP-1 consensus to mutant oligonucleotide; Lane 6 – Nuclear extract with 1:3 ratio of AP-1 consensus to mutant oligonucleotide; Lane 7 – Nuclear extract with 1:4 ratio of AP-1 consensus to mutant oligonucleotide; and

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

Important: Set the Odyssey Focus Offset to the center of the gel. For example, if the bottom glass plate is 1 mm thick and the gel 1 mm thick, set the focus offset to 1.5 mm.

LI-COR Biosciences


Quantification

One of the benefits of using Odyssey for EMSA analysis is that it provides an easy method for quantifica-tion. However, there are issues to consider when using Odyssey to quantify EMSA results. The primary issue is that the free DNA fragment has much less signal than the DNA fragment when bound to a protein, making quantification of the unbound DNA inaccurate. The addition of DTT/Tween to the binding reaction stabilizes the dye and reduces this phenomenon.

In addition, it is unrealistic to perform quantification analyses under the assumption that the free DNA band in the control, containing DNA only (no extract), should equal the sum of the signals of the free and bound DNA in the samples where the protein-DNA binding reaction occurs. Using end-labeled oligonucleotide duplexes as the DNA source and nuclear extract as a protein source renders this assumption impractical, due to the non-specific binding that occurs from using a nuclear extract. Oligonucleotides can also complicate quantification because the free oligonucleotides form a smear rather than a tight band. This makes it more difficult to assign an intensity value to bands.

III. Mobility Shift Experiment Example: p53 DNA Oligonucleotide Duplex to p53 Protein in Raji Nuclear Extract

Gel Preparation

Prepare 4% native polyacrylamide gel containing 50 mM Tris, pH 7.5; 0.38M glycine; and 2mM EDTA:

For 40 ml mix:

5 ml 40% polyacrylamide stock (Polyacrylamide-BIS ratio = 29:1)2 ml 1 M Tris, pH 7.57.6 ml 1 M Glycine160 µl 0.5 M EDTA26 ml H

2

O200 µl 10% APS30 µl TEMED

Pour the gel between glass plates and wait about 1-2 hours to polymerize.

Oligo Preparation

• p53 consensus oligonucleotides (5’-TAC AGA ACA TGT CTA AGC ATG CTG GGG-3’) were ordered from LI-COR, 5’ end-labeled with IRDye™ 700 phosphoramidites (Express Primers, Part #4200-31). These arrive as 1.0 nanomole dried powder.

• p53 mutant oligonucleotides (5’-TAC AGA ATC GCT CTA AGC ATG CTG GGG-3’) were ordered from LI-COR, 5’ end-labeled with IRDye™ 800 phosphoramidites (Express Primers, Part #4200-31B). These arrive as 1.0 nanomole dried powder.

1. Dilute oligos in 50 µl 1XTE for final concentration of 20 pmol/µl.

Warning: When quantifying Odyssey-generated EMSA data, great care must be taken in experimental design and analysis.



Binding Reaction

Volumes are in µl

* 100 mM TRIS, 500 mM NaCl, 10 mM DTT, pH 7.5** Add 1 µl of 1X binding buffer to the control reaction, instead of the nuclear extract.*** Raji extract was diluted to concentrations of 1.26 µg/µl, 1.69 µg/µl, 2.25 µg/µl, and 3 µg/µl in 1X binding buffer.

Electrophoresis

Odyssey Imaging

2. Place 5 µl of forward IRDye™ 700 labeled oligo into a new tube and add 5 µl of reverse IRDye™ 700 labeled oligo.

3. Place 5 µl of forward IRDye™ 800 labeled oligo into a new tube and add 5 µl of reverse IRDye™ 800 labeled oligo.

4. Anneal oligos by placing the oligo set in a 100°C heat block for 3 minutes. Leave the oligos in the heat block and turn it off to slowly cool to room temperature.

5. Dilute annealed oligos 1 µl in 199 µl water. This is your working DNA stock. Oligos can be stored at -20°C for up to a year if stored in darkness.

Reaction 1 2 3 4 5

10X Binding Buffer* 1 1 1 1 1Sterile ddH

2

O 4 4 4 4 425 mM DTT/2.5% Tween-20 2 2 2 2 2p53 oligo-IRDye™ 700 1 1 1 1 1p53 mutant oligo-IRDye™ 800 1 1 1 1 1Raji Nuclear Extract*** 0** 1 (1.26 µg) 1 (1.69 µg) 1 (2.25 µg) 1 (3 µg)

1. Order of addition: start with buffer, then add H

2

O, DTT/Tween, DNA and nuclear extract. After all reaction components are added, mix the reaction carefully.

2. Incubate at room temperature 20 minutes in darkness.

1. Add 1 µl of 10X Orange loading dye (LI-COR, #927-10100), mix, and load on a gel.

2. Run the gel at 10 V/cm for about 30 minutes in 1X TGE buffer.

1. Scan the gel inside the glass plates using 1.5 mm focus offset (assuming 1 mm thick gel and glass plates are 1 mm thick). If glass plates and gel are thicker/thinner, use larger/smaller offset (so that plane of focus is in the middle of the gel).

2. Start with Scan Intensity setting of 8 for both 700 and 800 channels.

LI-COR Biosciences


References

1. Wolf, S. S., Hopley, J. G., and Schweizer, M. (1994) The Application of 33P-Labeling in the Electro-phoretic Mobility Shift Assay.

Biotechniques

, 16, 590-592.

2. Suske, G., Gross, B., and Beato, M. (1989) Non-radioactive method to visualize specific DNA-protein interactions in the band shift assay.

Nucleic Acids Research

, 17, 4405.

3. Ludwig, L. B., Hughes, B. J., and Schwartz, S. A. (1995) Biotinylated probes in the electrophoretic mobility shift assay to examine specific dsDNA, ssDNA or RNA-protein interactions.

Nucleic Acids Research

, 23, 3792-3793.

2-Color Image

Figure 3. p53 EMSA using Raji Nuclear extract. (A copy of this document with color figures can be downloaded fromhttp://support.licor.com.)

0 1.26

µg

1.69

µg

2.25

µg

3 µg

Grayscale 700 nm Image

0 1.26

µg

1.69

µg

2.25

µg

3 µg

DNA Oligo

p53/DNA

Raji Nuclear Extract

Left, 2-color image shows the binding of the p53 consensus sequence (700 nm–red) and the p53 mutant non-specific binding (800 nm–green). Yellow color indicates the consensus sequence and mutant sequence are bound at the same location. Non-spe-cific binding is often seen when using nuclear extract as the protein source. Increasing the amount of the p53 mutant DNA will decrease or eliminate the inten-sity of the non-specific binding in the 700 nm channel.

Right, 1-color black and white view of the 700 nm image similar to an image that would be seen when using radioactively labeled DNA fragments. The 700 nm image only represents the p53 consensus binding complex and not the non-specific binding seen with 2-color analysis.

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LI-COR is an ISO 9001 registered company. © 2004 LI-COR Inc. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, Inc. Tween is a registered trademark of ICI Americas, Inc. The Odyssey Infrared Imager, IRDyes and the Odyssey system are covered by U.S. patents (6,495,812 and 6,593,148) and patents pending.

®

Doc# 988-08344

Odyssey


®

GEArray Q Series

Gene Expression Arrays

(SuperArray Biosciences Corp., Bethesda, MD


®

®

Odyssey

®

Protocol for GEArray Q Series Arrays


Contents

Page


II. Probe Synthesis................................................................................................1

III. Hybridization.....................................................................................................2

IV. Infrared Detection .............................................................................................3

V. Analysis ............................................................................................................4


Non-radioisotope GEArray

®

Q Series Gene Expression Array Kits can be purchased from SuperArray Bioscience Corp., Frederick, MD. For more information regarding array contents and availability, please contact SuperArray at 1-888-503-3187 or on-line at www.superarray.com.

These kits contain all instructions and components needed for GEArray

®

Q Series hybridization and detection except:

• RNA for probe generation• Biotin-16-dUTP (Roche, Cat. #1093-070) *• Sheared Salmon Sperm DNA (Invitrogen/Life Technologies, Cat. #15632-011)• 20X SSC• 20% SDS• IRDye™ 800CW-Streptavidin (LI-COR, Cat. #926-32230) or Alexa Fluor

®

680-Streptavidin (Invitrogen, Cat. #S-32358)

II. Probe Synthesis

There are three options for probe synthesis:

GEArray

®

RT-Labeling Kit (SuperArray, Cat. #L-01)

GEArray

®

TrueLabeling-RT Kit (TL-RT) Protocol (SuperArray, Cat. #L-02)

GEArray

®

AmpoLabeling-LPR Kit (SuperArray, Cat. #L-03).

Visit www.superarray.com for a detailed description of each of these labeling methods. Follow the SuperAr-ray manual for probe synthesis.


** IRDye™ 800CW-Streptavidin can also be purchased from Rockland Immunochemicals (Cat. #S000-31).

LI-COR Biosciences


III. Hybridization

Follow the SuperArray protocol for pre-hybridization, hybridization, and washing.

IV. Infrared Detection

V. Analysis

Spots on the arrays can be quantified using the Odyssey software grid feature with median background subtraction. Integrated intensity values can then be exported to an Excel spreadsheet for further analyses.

1. Block the array following the chemiluminescent detection protocol in the SuperArray manual.

2. Dilute 5X Buffer F five-fold to prepare a 1X Buffer F working solution.

3. Dilute IRDye™ 800CW-Streptavidin (1mg/ml) or Alexa Fluor

®

680-Streptavidin (1mg/ml) 1:7,500 with 1X Buffer F.

4. Remove GEA Blocking Solution Q from tube and add 2ml 1X Buffer F plus streptavidin.

5. Incubate for 10 minutes with gentle agitation in the dark at room temperature.

6. Wash according to SuperArray’s recommendation, followed by an additional 3-minute wash in 0.5% SDS to reduce background on the membrane.

7. Place array on the Odyssey Infrared Imaging System and scan at an initial intensity setting of 5. Intensity may need to be adjusted.

Protect from light during washes.

GEArray® Q Series Mouse Cell Cycle Gene Arrays hybridized with mouse brain (A), liver (B), or heart (C) total RNA detected using Alexa Fluor® 680-Streptavidin.

A. B. C.

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LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, inc. Alexa Fluor is a registered trademark of Invitrogen Corporation. GEArray is a trademark of SuperArray Bioscience Corporation. The Odyssey Infrared Imager and the Odyssey system are covered by U.S. patent (6,495,812), foreign equivalents, and patents pending.

®

Odyssey


®

®

Syto 60 Staining of Nucleic Acids in Gels

Doc# 988-08345


®

Syto

®

60 Staining of Nucleic Acids in Gels


Syto® 60 is a red fluorescent nucleic acid stain supplied as a 5 mM solution in DMSO by Invitrogen, catalog number S-11342. Any questions regarding Syto® 60 should be addressed to Invitrogen (www.invitrogen.com).

Syto

®

60 red fluorescent nucleic acid stain (5mM) was used to stain nucleic acids on 1.3% agarose gel and detected using LI-COR’s Odyssey

®

Infrared Imaging System. Sensitivity is high and background fluores-cence low. The detection limit for Syto 60 was determined to be 375 picograms for a 100 base-pair frag-ment (1.13 fmol), 95 picograms for a 500 base-pair fragment (0.6 fmol) and 44 picograms for a 1500 base-pair fragment (0.09 fmol). The detection limit for ethidium bromide using a Polaroid camera is 3000 pico-grams for a 100 base-pair fragment (90 fmol), 1500 picograms for a 500 base-pair fragment (9 fmol) and 700 picograms for a 1500 base-pair fragment (1.4 fmol). However, based on the more sensitive CCD imag-ing system, the detection limit of ethidium bromide is 750 picograms for a 100 base-pair fragment (23 fmol), 375 picograms for a 500 base-pair fragment (2.3 fmol) and 175 picograms for a 1500 base-pair fragment (0.4 fmol).

I. Introduction

Invitrogen’s patented Syto

®

Dyes are cell-per-meant cyanine dyes that bind to nucleic acids. Several Syto dyes are available with varying cell permeability, fluorescence enhancement upon binding to nucleic acids, excitation and emission spectra, and nucleic acid selectivity and binding affinity. Syto 60 has absorption and fluorescence emission maxima of 652/678 nm. Nucleic acids stained with Syto 60 can be detected and quantified on Odyssey using the 700 nm channel scan. In the presence of DNA, a ratio of 1 dye molecule to ~100 base pairs of nucleic acid is estimated.

Syto 60 was used to stain serial dilutions of a 100 bp DNA ladder (New England Biolabs, Catalog No. N3231S). Data are presented for Syto 60 staining and detection on Odyssey of DNA gels.

II. Methods

A serial dilution of a 100 base pair DNA ladder (New England Biolabs) from 1.0 microgram to 30 nano-grams was loaded onto 1.3% agarose, 1x TBE gels. Gels were electrophoresed at ~5-10 V per cm in 1x TBE running buffer, cut, and stained using various dilutions of Syto 60 in ddH

2

O. Various incubations at room temperature were evaluated. After staining, gels were rinsed briefly in distilled H

2

O and scanned on Odys-sey. Images were detected in the 700 nm channel. Table 1 summarizes the concentration versus time com-parison data.

The quickest staining time was 5 minutes using 1:1000 or 1:2000 dilution of Syto 60:H

2

O. Gels were stained sufficiently in 15 minutes using 1:2500 dilutions. A 1:5000 dilution of Syto 60 requires at least 30-45 minutes of staining. The most dilute solution tested was 1:20,000 and the gel was stained sufficiently after 45 minutes. There was no significant improvement in sensitivity from 60 to 120 minutes using 1:10,000, 1:15,000 and 1:20,000 dilutions.

LI-COR Biosciences


Table 1.

Dilution versus time comparisons including cost for a 25 ml staining solution (enoughto stain a gel about 10 x 10 cm and 5-8 mm thick.

III. Detection Limit

Up to 375 picograms of a 100 bp fragment (1.13 fmol), 95 picograms of a 500 bp fragment (0.6 fmol) and 44 picograms of a 1.5 kb fragment (0.09 fmol) can be detected using Syto 60 and Odyssey. The detection limit of ethidium bromide using a Polaroid camera is 3 nanograms for a 100 bp fragment (90 fmol), 1500 picograms for a 500 bp fragment (9 fmol) and 700 picograms for a 1.5 kb fragment (1.4 fmol). Based on the more sensitive CCD imaging system, the detection limit of ethidium bromide is slightly higher; 750 picograms for a 100 bp fragment (23 fmol), 375 picograms for a 500 bp fragment (2.3 fmol) and 175 picograms for a 1.5 kb fragment (0.4 fmol). Figure 1 shows a comparison between ethidium bromide and Syto 60.

Syto 60 DilutionAmount/25 mlSolution (µl) Cost per Gel

Minimum Time Needed for Staining (minutes)

1:1000 25 µl $5.50 5-151:2000 12.5 µl $2.75 5-151:2500 10 µl $2.20 15-301:5000 5 µl $1.10 30-45

1:10000 2.5 µl $0.55 451:15000 1.6 µl $0.35 451:20000 1.25 µl $0.28 45

Sensitivity = 4

1.5 KB

500 bp

100 bp

1.5 KB

500 bp

100 bp

Figure 1. Top Left: Image of ethidium bromide-stained gel using a Polaroid camera. Top Right: Image of ethidium bromide-stained gel using a CCD camera. Bottom: Scanned images of Syto®60-stained gel scanned on Odyssey, taken at varying sensitivities.

Odyssey: Syto 60 Odyssey: Syto 60 Odyssey: Syto 60

Polaroid: EtBr Gel CCD: EtBr Gel

Sensitivity = 3 Sensitivity = 5

Syto

®

60 Staining of Nucleic Acids in Gels


The appearance of speckles on the gel may be present after post-staining. Odyssey software’s "FILTER" function will improve the appearance of the scanned images (see Figure 2). To reduce the appearance of speckles on the gel, we recommend cutting off the wells before post-staining and rinsing the gel in running water.

NOTE:

The type and concentration of agarose will affect the degree of speckling. For example, low melting-point agarose tends to be highly prone to speckling.

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Figure 2. Image of agarose gel showing before and after using Odyssey software’s “filter” function.

Before Filter After Filter

®

Odyssey


®

Active Motif TransAM Transcription Factor Assays on the Odyssey System


™

®

®

Doc# 988-08346

TransAM

™

Transcription Factor Assays On The Odyssey

®

System


I. Introduction

Active Motif (Carlsbad, CA) offers a colorimetric/chemiluminescent ELISA based assay kit for transcription factor measurement. The assay is done in 96-well format broken down into 8-well strips. A wide range of assay kits are available (AP-1, p53, NF_B, CREB, etc.). For more information about the contents and avail-ability of these assays, please contact Active Motif at 1-877-222-9543 or www.activemotif.com. A specific oligonucleotide corresponding to the kit target protein has been immobilized to the bottom of a 96-well plate. Nuclear extract is bound to the DNA oligo followed by primary antibody incubation. To detect the protein of interest, a secondary HRP-conjugated antibody is applied and an HRP substrate is used. This gives a colorimetric/luminescent change that is measured. According to the protocol this takes less than five hours to complete. The nature of this assay makes it very amenable for use on the Odyssey

®

. A simple change from an HRP-conjugated secondary antibody to IRDye™-conjugated secondary antibody allows the assay to be converted for direct detection with Odyssey. This small change eliminates the enzymatic reaction steps in the protocol and reduces the amount of time needed to do the assay. It also eliminates the need for stopping the colorimetric reaction. The assay is easily completed in 3.5 hours. Dry assay plates can be stored in the dark and scanned at any time without compromising results.

II. Required Reagents

• TransAM™ Transcription Factor Assay Kit (Active Motif, Carlsbad, CA)• IRDye™ 800CW- conjugated antibody (Rockland Immunochemicals, Gilbertville, PA) which corre-

sponds to the HRP-conjugated antibody provided in the TransAM™ Transcription Factor Assay Kit.

III. General Guidelines for Converting the Colorimetric or Chemiluminescent Assay to an Infrared Assay

Binding of the Transcription Factor to the Consensus Sequence

Follow the protocol provided in the TransAM™ instruction manual.

Binding of the Primary Antibody

Follow the protocol provided in the TransAM™ instruction manual.

Binding of the Secondary Antibody

1. Add 100 µl of diluted IRDye™ 800CW conjugated secondary antibody (1:1000 dilution in 1X Antibody Binding Buffer) to all wells being assayed.

2. Cover the plate and incubate in the dark for 1 hour at room temperature without agitation.

3. Wash the wells 4 times with 200 µl 1X washing buffer (provided in kit).

LI-COR Biosciences


Detection


A TransAM™ p53 Assay Kit (Active Motif, Cat # 41196) was used with Raji nuclear extract dilutions of 15, 12.5, 10, 7.5, 5, 2.5, 1.25, and 0 µg. The protocol above was followed. Comparisons were made using complete binding buffer and complete binding buffer with free wild-type consensus oligonucleotide added to compete for binding (Figure 1.)

1. Place plate on the front left corner of Odyssey scanning surface. Scan the 800 channel using an initial intensity setting of 8, a resolution of 169 µm, and focus offset of 3 mm. If your image signal is saturated or too high, re-scan using a lower intensity setting. If your image signal is too low, re-scan using a higher intensity setting.

•

Figure 1. p53 TransAM data. Assay was done according to manufacturer’s protocol replacing the secondary HRP-antibody with a secondary IRDye™ 800CW antibody. A.) Odyssey image of the assay. Row 1 shows Raji nuclear extracts at dilutions of 15, 12.5, 10, 7.5, 5, 2.5, 1.25, and 0 µg of extract using complete binding buffer with free wild type consensus oligonucleotide. Row 2 shows Raji nuclear extracts of the same dilu-tions using complete binding buffer without competitor. B.) Data chart following negative control subtrac-tion as background.

Raji Extract with wild-type competitor Oligonucleotide

Raji Extract

A. B.

15 ng

12.5

10

7.5

5

2.5

1.25

0

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®

Odyssey


®

®

Adapting Panomics TranSignal Target Gene Arrays for the Odyssey System

Doc# 988-08347


™

®

Adapting TranSignal

™

Target Gene Arrays For The Odyssey

®

System


I. Introduction

Panomics, Inc. (Redwood City, CA) offers a variety of membrane expression array kits that can be adapted for use on the Odyssey

®

Infrared Imaging System. These arrays include:

For more information regarding array contents and availability, please contact Panomics, Inc. at 1.877.726.6642 or www.panomics.com.


Array Kit from Panomics, Inc.

Reagents used with Odyssey:

• Target Gene Array (2 each)• 20X SSC

• 10% SDS• 10 X Detection Buffer• Control RNA• Primer Mix• Distilled water• Labeling Mix• Biotin-dUTP• 10X Denaturing Solution• 2X Neutralization Solution

Reagents included in the kit but NOT used for infrared detection on the Odyssey System:

• Hybridization buffer• Luminol Enhancer• Stable Peroxide Solution• Streptavidin HRP conjugate• 10X Wash Buffer• 5X Blocking Buffer

Panomics, Inc. - Product Cat #

TranSignal™ Human p53 Target Gene Array

MA2010

TranSignal™ Mouse p53 Target Gene Array

MA2011

TranSignal™ Human NFkB Target Gene Array

MA2020

TranSignal™ Mouse NFkB Target Gene Array

MA2021

TranSignal™ Human Egr Target Gene Array

MA2030

TranSignal™ Mouse Interferon-Inducible Target Gene Array

MA2040

LI-COR Biosciences


Additional Reagents Required

• Total RNA/mRNA• AMV Reverse Transcriptase (Roche Molecular Biochemicals, Cat. #1495062)• UltraHyb™-OS (Ambion, Cat. #8662). This solution has been optimized for use with infrared probes.• Odyssey

®

Blocking Buffer (LI-COR, #927-40000)• IRDye™ 800CW-streptavidin (LI-COR, #926-32230 or Rockland Immunochemicals, Cat. #S000-31)• 1X PBST (0.1% Tween

®

-20)• 1X PBS• 20% SDS

III. General Guidelines for Converting the Chemiluminescent Assay to Infrared

Preparing Labeled cDNA Probes

Follow the protocol provided in the TranSignal™ Kit instruction manual for cDNA probe labeling.

Hybridization

Detection

1. Place array into a hybridization bottle. Wet membrane with dH

2

O and then decant the water.

2. Add 5ml of UltraHyb-OS (Ambion) Hybridization Solution to the bottle containing the array. Pre-hybridize at 42°C in a hybridization oven for 2 hours.

DO NOT use the hybridization solution provided in the array kit.

It causes very high levels of background on the Odyssey System.

3. Denature labeled-cDNA at 95°C for 5 minutes. Snap cool on ice.

4. Add the labeled-cDNA probe to 3ml of fresh UltraHyb-OS (Ambion) Hybridization Solution. Gently mix. Decant the pre-hybridization solution off of the array and pour labeled cDNA/hybridization solution into the bottle. Hybridize overnight at 42°C.

5. Decant the hybridization mixture from the hybridization bottle. Wash each membrane according to the protocol provided in the TranSignal™ Kit instruction manual.

1. Remove the membrane from the bottle using forceps. Transfer to a clean container with 5-10 ml (enough to cover the membrane) Odyssey Blocking Buffer plus 0.1% SDS (i.e. 2.5 ml 20% SDS in 500 ml Odyssey Blocking Buffer).

DO NOT use the blocking buffer provided in the array kit.

It causes very high levels of background on the Odyssey System.

Important: : DO NOT allow the membrane to dry during detection!

Adapting TranSignal

™

Target Gene Arrays For The Odyssey

®

System



■

See the protocol provided in the TranSignal™ Kit instruction manual

■

Arrays can be quantified using software included with the Odyssey Infrared Imaging System.

4308 Progressive Avenue • P.O. Box 4000 • Lincoln, Nebraska 68504 USATechnical Support: 800-645-4260 • North America: 800-645-4267 • International: 402-467-0700 • 402-467-0819


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LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey and IRDye are trademarks or registered trademarks of LI-COR, Inc. Ultrahyb is a trademark of Ambion Inc. TranSignal is a trademark of Panomics Inc. Tween is a registered trademark of ICI Americas, Inc. The Odyssey Infrared Imager, IRDyes and the Odyssey system are covered by U.S. patents (6,495,812 and 6,593,148), foreign equivalents, and patents pending.

2. Incubate at room temperature for 20 minutes with gentle shaking.

3. Add IRDye™ 800CW streptavidin (1mg/ml) to fresh Odyssey Blocking Buffer plus 0.1% SDS at a 1:1000 dilution (will need 3-5 ml to cover membrane).

DO NOT use the Streptavidin HRP conjugate provided in the array kit.

It cannot be visualized using the Odyssey System.

4. Decant the blocking buffer off the membrane and replace it with the IRDye™ 800CW streptavidin/Odyssey Blocking Buffer plus 0.1% SDS solution.

5. Incubate at room temperature in the dark for 20 minutes with gentle shaking.

6. Wash 3 times in 1X PBST (1X PBS + 0.1% Tween-20) for 5 minutes.

7. Rinse in 1X PBS for 5 minutes.

8. Scan wet membrane on the Odyssey instrument. Scan the 800 channel using an initial intensity setting of 8 and resolution of 84µm. If your image signal is saturated or too high, re-scan using a lower intensity setting. If your image signal is too low, re-scan using a higher intensity setting. Arrays can be quantified using Odyssey software.

®

Cartesian Array Human Cytokine Set 1

*

on the Odyssey System

* Biosource International, Camarillo, CA

Odyssey


®


™

®

®

Doc# 988-08348

Cartesian Array

™

Human Cytokine Set 1


I. Introduction

The Cartesian Array™ Human Cytokine Set 1 kit (Biosource International, Camarillo, CA; catalogue #BHM0011) is a protein assay that enables the simultaneous detection of 18 different cytokines in a single sample. With a few modifications, the Cartesian Array™ can be easily adapted for use on the Odyssey

®

Infrared Imaging System.

For detailed product information and availability please contact Biosource International at 1-800-242-0607 or visit www.biosource.com. For Odyssey support, contact LI-COR Biosciences at 1-800-645-4267.


Cartesian Array™ Kit Components

Components used with Odyssey:

• Four Cytokine Set 1 membranes• Biotin-Conjugated Anti-Cytokine Mix• 20X Wash Buffer• Eight-Well Incubation Tray• Manual

Components included in the kit but NOT used for infrared detection on the Odyssey System:

• 1,000X Streptavidin-HRP Conjugate• 1X Blocking Buffer• 20X Dilution Buffer• Detection Buffer A• Detection Buffer B


• Odyssey

®

Blocking Buffer (LI-COR Biosciences, Cat. #927-40000)• Tween

®

-20• IRDye™ 800CW-streptavidin (LI-COR, Cat. #926-32230*)• 1X PBS (Phosphate Buffered Saline)

* Streptavidin IRDye™ 800CW can also be purchased from Rockland Immunochemicals (Cat. #S000-31).

LI-COR Biosciences


III. Tips for Adapting the Cartesian Array Protocol for Detection on the Odyssey System

Best results can be obtained by following the instructions in the Cartesian Array Cytokine Array manual, with the following modifications:

■

Use Odyssey Blocking Buffer with 0.01% Tween-20

instead of

the Blocking Buffer referenced in the manual. Prepare blocking buffer by adding 25 µl of 20% Tween-20 to 50 ml of Odyssey Blocking Buffer; store at 4°C.

■

Use IRDye™ 800CW-streptavidin (for 800 channel detection)

instead of

the HRP-streptavidin provided with the kit. Optimal concentration can be determined empirically, but a starting concentration of 1.0 µg/ml will work well in most cases.

■

After incubation in dye-labeled streptavidin, membrane(s) should be washed

at least

four times in 1X Wash Buffer and two times with 1X PBS prior to imaging to insure there are no traces of dye remaining on the membrane(s). Use 2 ml of Wash buffer and 5 minutes for each wash.

■

Ignore the section in the Cartesian Array manual labeled “Detection Method”. After incubation in dye-labeled streptavidin and the subsequent washes, proceed directly to imaging on the Odyssey System.

■

Membranes should be scanned wet and care should be taken to avoid bubbles when placing them on the scanning surface. To prevent the membrane from drying out prior to imaging, store it in 1X PBS.

■

The following settings are recommended for imaging Cartesian Arrays on the Odyssey System and can be adjusted for best results:

• Resolution: 84 µm• Focus offset: 0.0 mm• Intensity: 3 (both channels)

IV. Final Notes

By comparing signal intensities, relative cytokine expression levels can be determined. Signal intensities can be quantitated using Odyssey application software or a favorite image/array analysis software package capable of importing 16-bit TIFF image files. For best assay results, include a negative control in which the sample is replaced with an appropriate mock buffer, especially when working with serum-containing media.

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®

Odyssey


®


RayBio Cytokine Antibody Arrays

*


* RayBiotech, Inc., Norcross, GA

®

®

®

Doc# 988-08349

RayBio

®

Cytokine Antibody Arrays


I. Introduction

RayBiotech, Inc. (Norcross, GA) offers a broad assortment of Human, Mouse, and Rat Cytokine Antibody Array kits, most of which can be easily adapted for use on the Odyssey

®

Infrared Imaging System. Both pre-selected and custom arrays are available in a simple membrane-based array format.

For a complete product listing, details, and availability please contact RayBiotech, Inc. at 1-888-494-8555 or visit www.raybiotech.com. For Odyssey support, contact LI-COR Biosciences at 1-800-645-4267.


RayBio

®

Kit Components


• Cytokine Array Membranes• Biotin-Conjugated Anti-Cytokines• 20X Wash Buffer I• 20X Wash Buffer II• 2X Cell Lysis Buffer*• Eight-Well Incubation Tray• Manual

* Only present in kits designated for cell lysate and certain tissue lysate samples.


• 2X Blocking Buffer• Detection Buffer A or C• Detection Buffer B or D• 20,000X HRP-Conjugated Streptavidin


• Odyssey

®

Blocking Buffer (LI-COR, Cat. #927-40000)• Tween

®

-20• IRDye™ 800CW-streptavidin (LI-COR, Cat. #926-32230)


LI-COR Biosciences


III. Tips for Adapting the RayBio Protocol for Detection on the Odyssey System

Best results can be obtained by following the instructions outlined in the RayBio Cytokine Array manual, with the following modifications:

■

Use Odyssey Blocking Buffer with 0.01% Tween-20

instead of

the Blocking Buffer referenced in the manual. Prepare blocking buffer by adding 25 µl of 20% Tween-20 to 50 ml of Odyssey Blocking Buffer; store at 4°C.

■


instead of


■


at least

four times in Wash Buffer I and two times with Wash Buffer II prior to imaging to insure there are no traces of dye remaining on the membrane(s). Use 2 ml of Wash buffer and 5 minutes for each wash.

■

Ignore the section in the RayBio manual labeled “Detection”. After incubation in dye-labeled streptavidin and the subsequent washes, proceed directly to imaging on the Odyssey System.

■

Membranes should be scanned wet and care should be taken to avoid bubbles when placing them on the scanning surface. To prevent the membrane from drying out prior to imaging, store it in Wash Buffer II.

■

The following settings are recommended for imaging RayBio arrays on the Odyssey System and can be adjusted for best results:


IV. Final Notes


4308




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LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, inc. Tween is a registered trademark of ICI Americas, Inc. RayBio is a registered trademark of RayBiotech, Inc. The Odyssey Infrared Imag-ing System is covered by U.S. patent (6,495,812), foreign equivalents, and patents pending.

®

Odyssey


®

Published October, 2007. The most recent version of this protocol is posted at http://biosupport.licor.com/support

Cayman Chemical* PPARγγγγ

Transcription Factor Kit Assay on the Odyssey System

* Cayman Chemical, Ann Arbor, MI

®

Doc# 988-09402

Cayman Chemical PPAR

γ

Transcription Factor Kit Assay


I. Introduction

The Cayman Chemical (Ann Arbor, MI) PPAR

γ

Transcription Factor Assay is a 96-well format ELISA-based kit for nonradioactive detection of DNA binding activity in nuclear extracts. Convenient 8-well strips are precoated with double-stranded DNA containing the peroxisome proliferator response element (PPRE) spe-cific for human peroxisome proliferator-activated receptors (PPAR) subtype

γ

. There is no cross-reactivity with the other PPAR subtypes

σ

or

α

. The PPARs in nuclear extracts bind the PPRE element and are detected using a primary antibody specific to PPAR

γ

. Binding of the secondary HRP-labeled anti-species conjugated antibody provides for colorimetric detection at 450nm. The kit includes buffer recipes and a protocol for purification of cellular nuclear extracts. Competitor DNA is provided as a means to confirm assay specific-ity. The employment of an HRP-conjugated detection antibody facilitates the conversion of the assay to a direct detection method using the Odyssey

®

system by substitution of IRDye

®

-labeled secondary antibody. This simple change reduces the amount of time to complete the assay and eliminates the need for addi-tional reagents to stop the reaction. Dry assay plates can be stored in the dark and scanned at any time without compromising results.


• PPAR

γ

Transcription Factor Assay Kit (Cayman Chemical, Cat. #10006855).• IRDye 800CW-conjugated antibody (LI-COR Biosciences) which corresponds to the HRP-conjugated

antibody provided in the Transcription Factor Assay Kit. For the PPAR

γ

kit use IRDye 800CW Goat Anti-Rabbit labeled secondary antibody (LI-COR Biosciences Cat. #926-32211).

• Odyssey Blocking Buffer containing 0.2% Tween

®

-20 (LI-COR Biosciences, Cat, #927-40000).

III. General Guidelines for Converting the Colori-metric Assay to an Infrared Assay

Binding of the Transcription Factor to the Consensus Sequence

Follow the protocol provided in the PPAR

γ

instruction manual.

Binding of the Primary Antibody

Follow the protocol provided in the PPAR

γ

instruction manual.

Binding of the Secondary Antibody

1. Add 100 µl of diluted IRDye 800CW-conjugated secondary antibody (1:1000 dilution in Odyssey blocking buffer containing 0.2% Tween-20) to all wells being assayed except blank wells.

2. Use the adhesive cover provided to seal the plate and incubate in the dark at room temperature for 1 hour without agitation.

3. Wash the wells 5 times with 200 µl of 1X wash buffer (provided in kit).4. Open the Odyssey cover, remove the strips from the plate carrier and place directly on the front left corner

of Odyssey scanning surface. Scan in the 800 channel using an initial intensity setting of 10, a resolution of

LI-COR Biosciences


169 µm, and focus offset of 1.5 mm. If the image signal is saturated, or too high, re-scan using a lower inten-sity setting. If the image signal is too low re-scan using a higher intensity setting.


Increasing amounts of positive control lysates supplied with the PPAR

γ

Transcription Factor Assay Kit were assayed for PPAR

γ

binding activity comparing the kit supplied HRP conjugated antibody to the IRDye 800CW conjugated secondary antibody (Figure 1A). A separate assay was performed in the presence of competitive ds DNA to confirm the specificity of the assay (Figure 1B). A reduction in signal correlates with binding of the competitive DNA rather than PPAR. The protocol above was followed. The data for the HRP detection method was collected using a Spectramax plate reader at 450 nm. The wells receiving the IRDye 800CW was scanned at intensity 10, 169 µm resolution and with a focus offset of 1.5 mm. The strip wells were placed directly on the scanner surface.

Figure 1. PPARγγγγ

Data. A.) Comparison of the data using increasing amounts (0, 5, 10, 15, and 20 µl) of pos-itive control lysate in complete binding buffer and either the HRP conjugated antibody or IRDye 800CW conjugated antibody for detection. B.) Comparison of reduction in signal with increasing amounts of added competitive DNA. Note that the increased sensitivity of the assay using the IRDye 800CW results in a more gradual drop-off in the signal with higher amounts of competitor, while the HRP detection signal drops sharply to 0 at both 5 and 10 µl of DNA.

A. Absorbance at 450 nm Odyssey Scan

B. Absorbance at 450 nm Odyssey Scan

4308




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®

Odyssey


®


TranSignal Cytokine Antibody Arrays

*


* Panomics, Inc., Redwood City, CA

™

®

®

Doc# 988-08350

TranSignal

™

Cytokine Antibody Arrays


I. Introduction

Panomics, Inc. (Redwood City, CA) offers a broad assortment of Human and Mouse Cytokine Antibody Array kits, most of which can be easily adapted for use on the Odyssey

®

Infrared Imaging System. Kits are available in a simple membrane-based array format; these arrays facilitate the simultaneous detection of multiple cytokines from a variety of sources, including cell lysates, certain tissue lysates, conditioned media, patient sera, plasma, and urine.

For a complete product listing, details, and availability please contact Panomics, Inc. at 1-877-726-6642 or visit www.panomics.com. For Odyssey support, contact LI-COR Biosciences at 1-800-645-4267.


TranSignal™ Kit Components


• Cytokine Array membranes• Biotin-Conjugated Anti-Cytokines• 20X Wash Buffer I• 20X Wash Buffer II• 1X Cell Lysis Buffer*• Eight-Well Incubation Tray• Manual

* Only present in kits designated for cell lysate and certain tissue lysate samples.


• 1,000X HRP-Conjugated Streptavidin• 1X Blocking Buffer• Detection Buffer A• Detection Buffer B


• Odyssey

®

Blocking Buffer (LI-COR, Cat. #927-40000)• Tween

®

-20• IRDye™ 800CW-streptavidin (LI-COR, Cat. #926-32230)


LI-COR Biosciences


III. Tips for Adapting the TranSignal Protocol for Detection on the Odyssey System

Best results can be obtained by following the instructions outlined in the TranSignal Cytokine Array manual, with the following modifications:

■

Use Odyssey Blocking Buffer with 0.01% Tween®-20

instead of

the Blocking Buffer referenced in the manual. Prepare blocking buffer by adding 25 µl of 20% Tween®-20 to 50 ml of Odyssey Blocking Buffer; store at 4°C.

■


instead of


■


at least

four times in Wash Buffer I and two times with Wash Buffer II prior to imaging to insure there are no traces of dye remaining on the membrane(s). Use 2 ml of Wash buffer and 5 minutes for each wash.

■

Ignore the section in the TranSignal manual labeled “Detection”. After incubation in dye-labeled streptavidin and the subsequent washes, proceed directly to imaging on the Odyssey System.

■

Membranes should be scanned wet and care should be taken to avoid bubbles when placing them on the scanning surface. To prevent the membrane from drying out prior to imaging, store it in Wash Buffer II.

■

The following settings are recommended for imaging TranSignal Arrays on the Odyssey System and can be adjusted for best results:


IV. Final Notes


4308




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LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey, and IRDye are trademarks or registered trademarks of LI-COR, inc. Tween is a registered trademark of ICI Americas, Inc. TranSignal is a trademark of Panomics, Inc. The Odyssey Infrared Imaging System is covered by U.S. patent (6,495,812), foreign equivalents, and patents pending.

®

®

Technical Note

Fluorophore-Linked Immunosorbent Assay (FLISA) Recommendations

Doc# 988-08352

Published January, 2006. The most recent version of this Technical Note is posted at http://biosupport.licor.com/support.

Notice:

LI-COR provides only limited tech-nical support for FLISA applications. Each FLISA assay will require individual optimi-zation and performance may vary.

Odyssey


®

FLISA Recommendations for Odyssey

®


The following should be used only as a guideline for adapting a Fluorophore-Linked Immunosorbent Assay (FLISA) to the Odyssey

®

Infrared Imaging System. LI-COR provides only limited technical support for FLISA (ELISA) application.

I. Assay Components

Microplates

Many commercially available microplates and strip wells designated for ELISA or EIA/RIA use are com-patible with Odyssey. A few recommended examples are given below. Consider the following charac-teristics when deciding to use a particular microplate: whether to use clear or opaque microplates, the diameter at the base of each well, and the distance from the bottom of the wells to the bottom of the supporting edge of the plate.

Clear vs. Opaque:

LI-COR recommends black microplates with optically clear bottoms to facilitate quantification. Clear microplates perform well, but produce significant laser light scatter around the well edges; extra care must be taken to exclude these edges during quantification.

Well Diameter:

It is necessary to know the well diameter for a particular microplate or 8-well strip; this diameter is used by the Odyssey application software for grid placement and determining well area for quantification. Well diameter can be determined by physically measuring the inside diameter of a well, searching the microplate manufacturer's specifications, or using the selection drawing tool within the application software (dimensions of a selected area are listed at the bottom of the window).

Focus Offset:

For microplates, the focus offset required in Odyssey application software is the distance from the inner surface at the base of the wells to the plate base that contacts the Odyssey scanning sur-face. Focus offset can be determined either physically, by measuring the distance (in millimeters); or empirically, by incrementing the focus offset over multiple scans of the same plate containing experi-mental or control samples. Quantify the same well, or wells, on each scan and determining the focus offset with the highest mean integrated intensity.

Microplate Examples:

■

Greiner Bio-One 96-well, polystyrene microplate; high binding capacityBlack with flat, optically clear well bottomsManufacturer's part number: 65509

■

Greiner Bio-One 384-well, polystyrene microplate; high binding capacityBlack with flat, optically clear well bottomsManufacturer's part number: 781097

■

Greiner Bio-One 96-well, polystyrene strip plate; 12 x 8-well removable strips; high binding capacityFlat, clear strips/wells in a microplate frameManufacturer's part number: 762071

■

Corning Costar 96-well, polystyrene microplate; high binding capacityBlack with flat, optically clear well bottomsManufacturer's part number: 3601

■

Corning Costar 96-well, polystyrene StripWell™ strip plate; 12 x 8-well removable stripsFlat, clear strips/wells in a microplate frame; high binding capacityManufacturer's part number: 2592

■

Corning Costar 96-well, clear polystyrene microplate; high binding capacityManufacturer's part number: 9018

LI-COR Biosciences


Suggested Reagents

Wash Buffers

■

1X PBS: This is commonly made from a 10X solution (LI-COR Biosciences Cat# 928-40018 or 928-40020) containing 1 M sodium phosphate and 1.5 M sodium chloride, but can also include potassium phosphate and/or potassium chloride depending on your preference. Check pH prior to use and adjust to 7.2 - 7.4 as necessary.

■

Alternate: 1x PBS + 0.05% Tween

®

-20 may be used when greater stringency is required for wash-ing. A final rinse with 1X PBS helps alleviate some of the frothing effect of the Tween-20. Take care when adding detergents to the wash buffer (e.g. SDS) as they can adversely affect other reagent components in the assay.

Blocking Buffer

■

1% BSA

■

5% Sucrose

■

0.05% Sodium Azide

■

Dillute in 1x PBS

■

Store at 4 °C

Alternate #1

0.1% Casein0.1% Tween-20 (optional)Dilute in 1X PBSStore at 4 °C

Alternate #2

Odyssey Blocking Buffer (LI-COR Biosciences Cat# 927-40000)0.1% Tween-20 (optional)

Note:

There are many other commonly used blocking reagents used for ELISA, each with its own advantages and disadvantages. If the aforementioned blocking buffers do not perform well in your system (i.e. the assay produces an exorbitant amount of nonspecific background signal), then you may want to experiment with other blocking agents and detergents.

Reagent Diluent

■

0.1% BSA

■

0.05% Tween-20

■

Dilute in 1X TBS (0.02M Tris base, 0.15M Sodium Chloride)

■

Adjust to pH 7.2 - 7.4 with HCl

■

0.2 µm filtered

■

Store at 4 °C

Alternatively, dilute with an appropriate blocking buffer or simply 1X PBS. Reagent diluent should only be used to dilute protein sample and detection antibody; dilute dye-labeled streptavidin and dye-labeled secondary antibody with 1X PBS.


®


Capture Buffer

■

15 mM sodium carbonate

■

35 mM sodium bicarbonate

■

0.02% sodium azide

■

Dilute in water

■

Store at room temperature

Detection Reagents

Streptavidin Conjugates

■

IRDye

®

800CW labeled Streptavidin (LI-COR Biosciences Cat# 926-32230)

Secondary Antibody Conjugates

■

IRDye 680 and 800CW labeled secondary antibodies are available from LI-COR Biosciences.

Protein Labeling Kits

■

IRDye 800CW Protein Labeling Kit (LI-COR Biosciences Cat# 928-38040, 928-38042, or 928-38044)

■

EZ-Link

®

Sulfo-NHS-LC Biotinylation Kit (Pierce Biotechnology Cat# 21430)

II. Optimization Considerations

Reagent Titration

Ideally, each reagent involved in the assay should be titrated in pairs; start by titrating the capture antibody and antigen, followed by titrating the detection antibody and secondary antibody or streptavidin conjugate. Realistically, however, the amount of antibody and/or antigen available may be a limiting factor in such an extensive titration. An example of a more efficient, though less thorough, titration of capture antibody, anti-gen, and detection antibody, is outlined in Figure 1. In addition, incubation temperatures and times can have significant effects on assay performance, and should be considered as part of the experimental design. In general, the temperature of incubation will determine the length of incubation time for each step in the assay. For example, standard/sample incubation performed at 4 °C will usually require an overnight incu-bation time, while incubation at 37°C will require a much shorter incubation time to obtain an equivalent fluorescent signal.

LI-COR Biosciences


Analyte

Standards:

A calibration curve, run in duplicate, should be generated for each experiment. Serial dilution range can be determined after an initial standard titration (see above) and should include a zero standard.

Samples:

If possible, samples should be filtered, column purified, dialyzed, or otherwise purified for best results. Dilute samples in Reagent Diluent (recommended), blocking buffer, or 1X PBS.

Fluorescent Detection

The recommended method of detection for FLISA assays is biotin/streptavidin because many commer-cially available ELISA kits use biotinylated detection antibodies along with streptavidin-conjugated horseradish peroxidase or alkaline phosphatase. This method allows you to purchase an ELISA kit and substitute the HRP/AP-streptavidin with a fluorescent IRDye-labeled streptavidin.

For cases in which a biotin conjugated detection antibody is either unavailable or undesirable, an alter-native detection option would be to utilize a dye-labeled secondary antibody against an unlabeled detection antibody; however, this format generally does not provide the level of sensitivity of biotin/streptavidin. Another detection alternative is to employ a dye-labeled antibody conjugate directly; this option has the advantage of requiring one less reagent, but may introduce additional problems with non-specific fluorescent signal.

654321 121110987

Zero StandardA

B

C

D

E

F

G

H

Standard #1

Standard #2

Standard #3

Zero Standard

Standard #1

Standard #2

Standard #3

#3 #3#2#2 #1#1

Detection Antibody Concentration

Capture Antibody Concentration #3 Capture Antibody Concentration #4

Capture Antibody Concentration #1 Capture Antibody Concentration #2

Figure 1. Example of a plate outline for reagent titration. Each quadrant of this par-ticular plate tests three different detection antibody concentrations, each with four standard concentrations in duplicate (including a zero standard). These titrations are repeated in each quadrant for four di stinct capture antibody concentrations. Anti-body/streptavidin conjugate is maintained at a fixed concentration.


®


Wash Procedure

The wash steps in the FLISA assay are critical to obtaining good results. Use the following guidelines for best results:

■

Standard laboratory wash bottles work well for washing. Automatic plate washers may be used when higher throughput is necessary, as long as cross-contamination between wells can be pre-vented and the guidelines below can be adhered to. Wells can be overfilled and sprayed vigor-ously, but not harshly. Make sure each well is filled completely with buffer for each wash (exception: for the last set of washes, use a pipet to dispense and aspirate wash buffers. This will prevent extraneous signal caused by dye conjugate spilling over between wells.).

■

For more stringent washing, first wash 2-3 times with 1X PBS-T, then rinse with 1X PBS. After the final wash, briefly centrifuge the plate upside-down on a clean paper towel at low speed (~600 x g); this will ensure all liquid is removed and allow for better reagent contact in the microplate wells for the successive steps. If a centrifuge is not available, rigorously (but not harshly) blot the plate on paper towels until all liquid is removed from the wells.

■

Optionally for each wash, allow the wash buffer to sit in the wells for 1-2 minutes with gentle rocking/shaking.

III. Odyssey Settings

Scan

Start by defining a Scan Preset to use with the FLISA assay. Choose

Settings > Scan Presets

in Odyssey application software and edit the default “MicroPlate2” scan preset. Change the focus offset to match the distance measured from the well bottom to the base of the plate. Click

Save As

and save the Scan Preset using a new name. Consult the Odyssey User Guide for complete information on using the Scan Preset settings.

Sample Quantification

Quantification in Odyssey application software is performed by applying a grid to the microplate image (choose

Analyze > Add Grid

). If one of the default Grid Templates does not fit your microplate, use the Grid Template settings (choose

Settings > Grid Templates

) adjust the well diameter and other parame-ters to correspond with the microplate you are using. Quantification data can be viewed in the grid sheet (choose

Analyze > Grid Sheet

). Consult the Odyssey User Guide for more detailed microplate quantification procedures.

LI-COR Biosciences


IV. General FLISA Protocol

The following is an example protocol for performing a 96-well microplate sandwich FLISA with the Odys-sey Infrared Imaging System. Reagent concentrations, incubation times and temperatures, and method of fluorescent detection are given only as a typical FLISA example; these characteristics may vary consider-ably with the samples and antibodies in your assay system.

1. Bring all reagents to room temperature before use.

2. Prepare an appropriate amount of 2 µg/ml capture antibody. Pipet 100 µl into each desired well. Incubate microplate at 37 °C for 30 minutes, room temperature for 2 hours, or 4 °C overnight (16-18 hours). Gently agitate plate on a shaker or rocker during incubation.

3. Remove capture antibody solution from wells. Wash 3 times with 1X PBS-T and once with 1X PBS; use enough wash buffer to fill each well. Gently agitate wash buffer for 1-2 minutes before removing. Ensure wash buffer is removed completely before proceeding to the next step.

4. Pipet 300 µl of blocking solution into each well. Incubate at room temperature with gentle agitation for at least one hour. Prepare samples and standards while waiting for blocking to proceed.

5. Remove blocking buffer completely.

6. Add 100 µl of the appropriate samples/standards dilutions to each well. Incubate at 37 °C for 30 minutes, room temperature for two hours, or 4 °C for 4-6 hours. Gently agitate plate during incubation.

7. Remove samples/standards from wells. Wash 3 times with 1X PBS-T and once with 1X PBS as in step (3).

8. Prepare an appropriate amount of 200 ng/ml biotinylated detection antibody. Pipet 100 µl into each well. Incubate at 37 °C for 30 minutes, room temperature for 1 hour, or 4 °C for 2-3 hours.

9. Remove detection antibody solution from wells. Wash 3 times with 1X PBS-T and once with 1X PBS as in step (3).

10. Prepare an appropriate amount of 1 µg/ml streptavidin conjugate. Add 100 µl to each well. Protect the plate from light and incubate at room temperature with gentle agitation for 30 minutes.

11. Carefully decant the streptavidin solution with a pipet. Wash 4 times with 1X PBS-T and once with 1X PBS; take extra care during this set of wash steps so as not to allow wash buffer to spill between wells (and thereby introduce a potential source for extraneous fluorescent signal).

12. Place the microplate on the Odyssey scanning surface with well A1 in upper left orientation. Use the microplate scanning guide as described in the Odyssey Operator’s Manual. Scan the microplate following the aforementioned guidelines and instructions in the Odyssey User Guide.

4308




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®

Odyssey


®

®

Technical Note

Tips for Antibody and Lysate Arraying

Doc# 988-08014

Published May, 2005. The most recent version of this Technical Note is posted at http://biosupport.licor.com/support.

Tips For Antibody And Lysate Arraying


I. Tips For Antibody Arraying

See V&P Scientific, Inc. “Technical Note 42” for a detailed description of the care and use of the Manual Glass Slide Microarrayer.

Antibody Arraying

1. Getting Started

�

Antibody arraying can be done at room temperature.

�

If desired, a cooling pack can be placed underneath the source plate to keep source solutions cold.

�

Humidity needs to be 50-70% during arraying to prevent evaporation from source plate.

2. Antibody Dilution

�

Dilute capture antibodies in 1X PBS.

�

Suggested capture antibody concentration is 0.1-1mg/ml.

�

20 µl per source plate well is needed.

�

Use a V-bottom 96-well plate, such as NUNC™ polystyrene 96-well V bottom plate (VWR catalogue No. 73520-486) as source plate.

3. Slide Arraying

�

The mechanics and methodology of arraying using the Manual Glass Slide Microarrayer can be found in V&P Scientific, Inc. “Technical Note 42”.

�

When using nitrocellulose-coated slides, it is suggested that every other vertical indexing hole is used, rather than all 12. If all 12 vertical index holes are used, spots may spread into each other. All 8 horizontal indexing holes can be used.

Note: Although the pin diameter is 457 µm, the spot size can be considerably larger because liq-uid spreads on nitrocellulose. Spot size depends on the nature of the source solution. Spot pitch is 750 µm on the vertical axis and 1,125 µm on the horizontal axis.

�

After printing is complete, let slides air dry for 1 hour.

Assay And Detection

1. Blocking

�

Block in Odyssey

®

Blocking Buffer for 1 hour at room temperature on a shaker with gentle agitation.

Note: Optimization of blocking buffer will be needed for best antibody performance.

�

The incubation can be done in a plastic bag, hybridization chamber, or box.

2. Sample Binding

�

Add protein sample diluted in Odyssey Blocking Buffer.

�

Incubate on an orbital shaker at RT for 1-2 hours or overnight at 4°C.

3. Wash

�

Wash 3 times with 1X PBST (0.1% Tween-20

®

), 5 minutes per wash.

LI-COR Biosciences


4. Detection Using Biotinylated Detection Antibody

�

Make a 1:500 dilution of biotinylated detection antibody in Odyssey Blocking Buffer.

�

Incubate at RT for 1-2 hours on an orbital shaker.

Note: The detection antibody dilution must be optimized. A 1:500 dilution is a suggested starting point.

�

Incubate the slides in diluted antibody for 1-2 hours at room temperature on an orbital shaker.

�

Wash 3 times with 1X PBST (0.1% Tween-20), 5 minutes per wash.

�

Make a 1:5000 dilution of 1mg/ml streptavidin-IRDye™ 800CW (LI-COR, Part # 926-32230) in Odyssey Blocking Buffer plus 0.1% Tween-20.

�

Incubate the slides in diluted IRDye™ 800CW-streptavidin for 30 minutes at room temperature on a shaker in the dark.

�

Wash 3 times with 1X PBST (0.1% Tween-20), 5 minutes per wash.

�

After the last wash, scan the wet slide to see if the washing was sufficient (background is high with insufficient washing). If so, dry the slide by centrifugation. If not, wash one more time.

TIP:

If a slide centrifuge is not available, the slide can be placed into a 50 ml conical tube and centrifuged at a low RPM for drying.

�

Scan the slide on the Odyssey scanner with 42 µm resolution and an initial intensity setting of 5. If the signal is too strong or weak, rescan at a lower or higher intensity setting.

II. Tips For Lysate Arraying (Reverse Phase Array)

See V&P Scientific, Inc. “Technical Note 42” for a detailed description of the care and use of the Manual Glass Slide Microarrayer.

Cell Lysate Arraying

1. Getting Started

�

Cell lysate arraying can be done at room temperature.

�

If desired, place a cooling pack underneath the source plate to keep the source solutions cold.

�

Humidity needs to be 50-70% during arraying to prevent evaporation from source plate.

2. Cell Lysate Preparation

�

Use appropriate lysis buffer to prepare cell lysates.

Note: The lysis buffer MUST NOT contain any loading dye; loading dyes may be detected by the Odyssey.

�

Boil lysates for 5 minutes, centrifuge at 14,000 rpm for 2 minutes, and store at 4°C.

�

Immediately before arraying, transfer lysates to a source plate with two-fold serial dilutions to determine the best concentration for future arraying.

�

20 µl per source plate well is needed.

■

Use a V-bottom 96 well plate, such as NUNC™ polystyrene 96 well V bottom plate (VWR cata-logue No. 73520-486) as source plate.

3. Slide Arraying

■

The mechanics and methodology of arraying using the Manual Glass Slide Microarrayer can be found in V&P Scientific, Inc. “Technical Note 42”.

■

It is suggested that every other vertical indexing hole is used rather than all 12. If all 12 vertical index holes are used, spots may spread into each other. All 8 of the horizontal indexing holes can be used.

Note: Although the pin diameter is 457 µm, the spot size can be considerably larger because liquid spreads on nitrocellulose. The spot size depends on the nature of the source solution. The spot pitch is 750 µm on the vertical axis and 1,125 µm on the horizontal axis.

■

After printing is complete, let the slides air dry for 1 hour.

Assay And Detection

Note: It is highly recommended that the primary antibodies used for lysate array detection be validated by Western Blot analysis using the identical blocking and detection methods.

1. Blocking

■

Block in Odyssey Blocker for 1 hour at room temperature on a shaker with gentle agitation.

NOTE: Optimization of blocking buffer will be needed for best antibody performance.

■

The incubation can be done in a plastic bag, hybridization chamber, or box.

2. Primary Antibody Binding

■

Make a 1:500 dilution of primary antibody in Odyssey Blocking Buffer plus 0.1% Tween-20.

Note: The Primary antibody dilution needs to be optimized. A 1:500 dilution is a suggested starting point.

■

Incubate the slides in diluted antibody on an orbital shaker for 0.5-2 hours at room temperature or overnight at 4°C.

3. Wash

■

Wash 3 times with 1x PBST (0.1% Tween-20), 5 minutes per wash.

4. Detection

■

Make a 1:5000 dilution of 1mg/ml Secondary Antibody labeled with IRDye™ 800CW in Odyssey Blocking Buffer plus 0.1% Tween-20.

■

Incubate slides in diluted antibody for 1 hour at room temperature on a shaker in the dark.

■

Wash 3 times with 1x PBST (0.1% Tween-20), 5 minutes per wash.

■

After the last wash, scan the wet slide to see if the washing was sufficient. If so, dry the slide by centrifugation. If not, wash one more time.

TIP:

If a slide centrifuge is not available, the slide can be placed into a 50ml conical tube and centrifuged at a low RPM for drying.

■

Scan the slide on the Odyssey scanner with 42 µm resolution and an initial intensity setting of 5. If the signal is too strong or weak, rescan at a lower or higher intensity setting.

LI-COR Biosciences • 4308

Progressive Avenue • P.O. Box 4000 • Lincoln, Nebraska 68504 USATechnical Support: 800-645-4260 • North America: 800-645-4267 • International: 402-467-0700 or 402-467-0819LI-COR GmbH (Germany, Austria, Switzerland, Czech Republic, Hugary, Slovakia): +49 (0) 6172 17 17 771LI-COR UK Ltd.: +44 (0) 1223 422104

LI-COR is an ISO 9001 registered company. © 2005 LI-COR Inc. LI-COR, Odyssey and IRDye are trademarks or registered trademarks of LI-COR, Inc. Tween is a registered trademark of ICI Americas, Inc. Nunc is a trademark of Nunc A/S Corporation. The Odyssey Infrared Imager and the Odyssey system are covered by U.S. patent 6,495,812, foreign equivalents, and patents pending.

Odyssey


®

®

Technical Note

Scanning a Mouse on Odyssey: Hints and Tips

Doc# 988-08080

Published September, 2005. The most recent version of this Technical Note is posted at http://biosupport.licor.com/support.

Notice and Disclaimer:

LI-COR Biosciences provides only limited technical support for imaging mice or other small animal species on the Odyssey Infrared Imaging System. The enclosed recommendations are provided “as is,” for informational purposes only. LI-COR makes no warranty of any kind with regard to this written material or imaging appli-cation. User/purchaser is responsible for adhering to all applicable institutional, state, and federal guidelines for the care and use of research animals.

Scanning a Mouse on Odyssey: Hints and Tips


I. Diet Considerations

Mouse chows generally have high fluorescent signal in the 700- and 800-channels due to plant based ingredients (ie, chlorophyll). If imaging in the abdominal region where intestinal fluorescence will be an issue, feed a purified diet containing no plant based ingredients or a less expensive alternative would be to fast the animals over night. Figure 1 illustrates the issues with regular mouse chows (signal is saturated) as compared to two purified diets provided by the same company.

An example of the level of interference that can be seen when imaging a mouse on Odyssey is shown in Figure 2. The circle on the 700 channel image indicates the intestinal signal likely due to the mouse diet while the circle on the 800 channel image pin-points the abdominal tumor.

Harlan Teklab

LM-485

Figure 1. Three mouse chows from Harlan Teklab imaged on Odyssey.

700 Channel

800 Channel

Harlan Teklab

AIN-93GHarlan Teklab

AIN-93M

Figure 2. A typical mouse scan on Odyssey where an abdominal tumor was present. Scan parameters include resolu-tion = 169 µm; quality = medium; focus offset = 2.0; intensity values = L1 for 700 channel and 3 for 800 channel.

800 Image Only 700 Image Only

Abdominal Tumor

LI-COR Biosciences


II. Pre-Scan

It is always beneficial to scan the mouse prior to probe injection to document the amount of background/autofluorescence the mouse emits. Start with intensity setting of L1 and 3 for the 700 and 800 channel, respectively, until the level of expected signal is known.

III. Resolution

Your first image should be at the lowest resolution (ie, 337) which gives a good preview of what to expect from the particular mouse model being evaluated. The short pre-scan will provide a more accurate esti-mate of what intensity settings should be if the mouse is positioned optimally. Generally, images scanned at 169 µm are a good compromise between resolution and scan time, however, there is no reason not to scan at higher resolution, such as 21 µm, just expect the scan time to increase dramatically depending on the size of the scan area.

IV. Hair vs. Shaved Animal

If working with a haired mouse model (ie, something other than nude mice), shave the animal in the region of interest prior to imaging. Up to 50% of the signal is blocked when imaging through hair. Hair removal can be accomplished by shaving (ie, mustache shavers work well) or by the use of Nair

®

. To demonstrate, a mouse was implanted with a tube containing IRDye™ 800CW in the thoracic cavity and imaged before and after shaving. Figure 3 shows the effect of hair with the difference between Panel A (before) and Panel B (after) being 53%.

V. Reflections

Any areas that may cause a reflection will be an issue. For example, when shaving, avoid nicking the skin as the open nick will cause reflection and signal.

Figure 3. Signal from IRDye™ 800CW in the thoracic cavity when imaged on Odyssey before (panel A) and after (panel B) shaving.

A. B.

VI. Focus Offset for Surface and Abdominal Tumors

A focus offset of 1-2 mm is a good starting point for a surface (subcutaneous) tumor. If the tumor is intra-abdominal then a focus offset of 4.0 mm should be the starting point.

LI-COR Biosciences • 4308

Progressive Avenue • P.O. Box 4000 • Lincoln, Nebraska 68504 USATechnical Support: 800-645-4260 • North America: 800-645-4267 • International: 402-467-0700 or 402-467-0819LI-COR GmbH (Germany, Austria, Switzerland): +49 (0) 6172 17 17 771 • LI-COR UK Ltd.: +44 (0) 1223 422104

LI-COR is an ISO 9001 registered company. © 2005 LI-COR Inc. LI-COR, Odyssey and IRDye are trademarks or registered trademarks of LI-COR, Inc. Nair is a registered trademark of Church & Dwight Co., Inc. The Odyssey Infrared Imager and the Odyssey system are covered by U.S. patent 6,495,812, foreign equivalents, and patents pending.

Figure 4. Subcutaneous tumor (800 channel) and liver and cecum signal present in the 700 channel.

800 Image Only 700 Image Only

Subcutaneous Tumor

Liver Signal

Cecum Signal

Figure 5. Abdominal tumor in the 800 channel and intestinal signal in the 700 channel.

800 Image Only 700 Image OnlyAbdominal Tumor

Figure 6. The 800-channel image illustrates clearance of the particular IRDye™ 800CW labeled probe from the kidneys. A subcutaneous side tumor is also visible.

Subcutaneous TumorKidneys

®

IRDye 800CW Protein Labeling Kit - High MW

Published August, 2006. The most recent version of this protocol is posted at http://biosupport.licor.com/support

®

IRDye

Infrared Dye Reagents

®

Part Number: 928-38040

IRDye

®

800CW Protein Labeling Kit - High MW

Page 1

Contents

Page

I. Introduction .......................................................................................................1II. Kit Components ................................................................................................2III. Preparation of Protein Solution for Conjugation ...............................................2IV. Protein Labeling Reaction.................................................................................3V. Separation of Conjugate from Free Dye ...........................................................4VI. Calculation of Dye/Protein Ratio and Protein Concentration ............................4VII. Handling of Labeled Conjugates.......................................................................6VIII. Troubleshooting Guide......................................................................................6

IX. Reference .........................................................................................................8

I. Introduction

The IRDye

®

800CW Protein Labeling Kit - High MW is optimized to label proteins for use with the Odyssey

®

Infrared Imaging System, Aerius™ Automated Imaging System, or other

in vivo

imaging systems with near-infrared detection. Labeled proteins may be used for Western blots, In-Cell Westerns (ICW),

in vivo

imaging, and other applications.

The kit is optimized for labeling 1 mg of protein with molecular weight 45 - 200 kDa. For proteins of lower molecular weight, use IRDye 800CW Protein Labeling Kit - Low MW (P/N 928-38042). For small amounts of protein (100 µg) with molecular weight 14-200 kDA, use IRDye 800CW Protein Labeling Kit - Microscale (P/N 928-38044).

The IRDye 800CW dye bears an NHS ester reactive group that will couple to proteins and form a stable conjugate. Fluorescent conjugates labeled with IRDye 800CW display an absorption maximum of 774 nm and an emission maximum of 789 nm in 1X PBS (Figure 1). These spectral characteristics match the 800 nm channel on the Odyssey and Aerius.

Figure 1. Absorption and emission spectra of IRDye 800CW in 1X PBS.

LI-COR Biosciences

Page 2

II. Kit Components

• 3 x IRDye 800CW Reactive Dye vials (0.1 mg). Store at -20°C.• 1 x 0.5 mL 1M Potassium Phosphate (K

2

HPO

4

), pH 9 (store at 4°C)• 1 x 25 mL 1X PBS (store at 4°C)• 1 x 0.5 mL ultra pure water (store at 4°C)• 3 x Pierce

®

Zeba™ Desalting Spin Columns, Product 89891 (store at 4°C)

Note:

The minimum recommended protein molecular weight for these columns is 7 kDa. • Pierce Zeba™ Desalting Spin Column instructions • Protocol for IRDye 800CW Protein Labeling Kit - High MW

III. Preparation of Protein Solution for Conjugation

To perform a labeling reaction it is critical for the protein to be in a preservative-free phosphate buffer at pH 8.5. Preservative (i.e. sodium azide) removal and buffer exchange can be accomplished by passing the protein through a desalting column similar to those provided in the kit (additional columns not included). For buffer exchange, the column should be equilibrated with 50 mM phosphate buffer, pH 8.5. Alterna-tively, the protein solution can be dialyzed (cassette not included) against 50 mM phosphate buffer, pH 8.5. If the protein solution is free of preservatives and in a buffer with a pH lower than 8.5, the pH of the solu-tion can be raised by adding the concentrated phosphate buffer (1M potassium phosphate, pH 9) included in the kit to the protein solution.

1. Prepare 1.0 mg of protein in a phosphate buffer without sodium azide, at a concentration of 1 (± 0.1) mg/mL.

Notes:

• Protein concentration can be determined spectrophotometrically using the extinction coefficient of the protein or colorimetrically using several kits (e.g. Pierce BCA) which are commercially available. If using a colorimetric analysis, the standards must be prepared in the same protein as the samples to be measured. BSA does not provide a representative standard curve for all proteins.

• Use of a more dilute or more concentrated protein solution will result in decreased or increased labeling of the protein, respectively.

• The protein to be labeled must be in a buffer that is free of primary amines and ammonium ions. Tris or glycine buffers cannot be used for conjugation. Even trace amounts of components containing primary amines will decrease labeling efficiency.

• Unpurified antibodies (such as ascites fluid and crude serum), cell lysates, and proteins that contain BSA or other proteinaceous stabilizers will not label well and have not been characterized for use with this kit.

2. Raise the pH of the preservative-free protein solution to pH 8.5 with the 1M Potassium Phosphate buffer (K

2

HPO

4

), pH 9, provided in the kit, as necessary. If the protein solution is in 1X PBS adjust the pH by adding 1/10

th

volume of 1M Potassium Phosphate buffer, pH 9. For example, for 1.0 mL of protein solution, add 0.1 mL of 1M Potassium Phosphate buffer, pH 9.

3. Cool/warm the protein to 20-25 °C before reaction with the dye.

Note:

• If the protein is temperature sensitive, the labeling reaction may be carried out at a lower temperature.

IRDye

®


Page 3

IV. Protein Labeling Reaction

1. Use Figure 2 to determine the appropriate amount of dye to add based on the molecular weight of your protein.

Notes:

• Using the dye amounts shown in Figure 2 typically results in a D/P ratio of 1:1 to 3:1.

• The optimal degree of labeling will vary for different applications, and the effect of labeling on the biologi-cal activity of the protein will depend on factors including size and amino acid composition. Over-labeling may cause high background or self-quenching of the dye.

• Due to variation in amino acid composition, different proteins will react with the dye at different rates. It may be necessary to adjust the standard protocol to achieve optimal labeling.

• A D/P ratio of 1:1 - 2:1 for an IgG antibody is suitable for both Western and In-Cell Western applications. Higher D/P ratios (3:1 - 4:1) for an IgG antibody may still be usable for Western blot detection, but may exhibit increased background and therefore not perform optimally for other applications.

• For

in vivo

imaging applications, the dye/protein ratio of the conjugate may affect biological or biochemical activity of the protein, signal-to-noise ratio, blood clearance, and biodistribution (for example, Schellen-berger

et al.

, 2004).

2. Dissolve 1 tube of dye with 25 µl of ultra pure water provided in the kit.

Note:

• Work quickly as the dye reactivity decreases over time.

3. Mix the appropriate amount of dye with 1.0 mg of protein. React for 2 hours at 20°C, protecting the vial from light.

Note:

• Little difference in labeling efficiency was noted from 4-25°C. However, the kit is optimized for reactions at 20°C. If the labeling reaction is performed at temperatures less than 20°C, any remaining reactive dye must be removed from the sample immediately after the 2 hour incubation (see Section V). At 20-25°C, there is little or no reactive dye remaining in the reaction mix after 2 hour incubation.

4. Separate the free dye from the protein conjugate as outlined in Section V below.

Dye Volume= 1166 MW

0.0

5.0

10.0

15.0

20.0

25.0

0 50 100 150 200 250

MW (kDa)

Dye

Vo

lum

e (

L)

5.83200

7.29160

11.7100

14.680

17.965

21.255

25.045

(µL)(kDA)

Dye VolMW

Figure 2. Suggested dye amounts based on protein molecular weight.

LI-COR Biosciences

Page 4

V. Separation of Conjugate from Free Dye

Detailed instructions for use of the Pierce Zeba™ Desalting Spin Columns are included with this kit. Instructions can also be downloaded from www.piercenet.com.

The Pierce Zeba™ Desalting Spin Columns are suitable for 0.5 - 2.0 mL of reaction volume. Other sizes are available from the manufacturer. The recommended molecular weights for the spin columns are > 7 kDa.

VI. Calculation of Dye/Protein Ratio and Protein Concentration

Once the free dye has been sufficiently removed, the dye to protein ratio of the conjugate can be deter-mined. Calculate the number of dye molecules per protein molecule (dye/protein or D/P ratio) by measur-ing absorbance with a UV-Vis spectrophotometer.

Important:

When the protein is highly labeled with IRDye 800CW, the absorption spectrum in 1X PBS shows a strong “blue” shoulder (~705 nm) near the normal dye peak. This distortion will cause the calculated D/P ratio to be lower than the true ratio. To correct for this, always dilute the conjugate in a solvent mixture of 1X PBS and Methanol (1:1) to determine the dye/protein ratio.

1. Read and follow instructions for use of Pierce Zeba™ Desalting Spin Columns before proceeding.

Note:

• Never reuse the Pierce Zeba™ Desalting Spin Columns.

2. Remove the 0.05% azide preservative from the column by following the Pierce Zeba™ Desalting Spin Column “Procedure for Buffer Exchange” using the 1X PBS supplied in the kit.

3. Use the Pierce Zeba™ Desalting Spin Column to purify the dye labeled conjugate.

Note:

• Dye labeled conjugates obtained following the Pierce Zeba™ Desalting Spin Column protocol typically contain 5% or less free dye.

1. Dilute the labeled conjugate 1:10 to 1:50 in a mixture of 1X PBS and Methanol (1:1) such that the maximum absorbance reading at A

780

is less than 2.0 and the A

280

is greater than 0.12.

2. Measure the absorbance of the conjugate at 280 nm and 780 nm (A

280

and A

780

).

IRDye

®


Page 5

3. Calculate the dye/protein ratio using this formula:

In which:

■

0.03 is a correction factor for the absorbance of IRDye 800CW at 280 nm (equal to 3.0% of its absorbance at 780 nm).

■

ε

Dye

and

ε

Protein

are molar extinction coefficients for the dye and protein, respectively.

■

ε

Dye

is 270,000 M

-1

cm

-1

and

ε

Protein

is 203,000 M

-1

cm

-1

(for a typical IgG) in a 1:1 mixture of PBS:Methanol.

Proteins other than IgG may have very different molar extinction coefficients; use of the correct extinction coefficient for your protein is essential for accurate determination of the D/P ratio.

4. Calculate the final protein concentration using this formula:

In which:

■

MW

Protein

is the molecular weight of the protein.

■

Dilution factor is the dilution of the labeled conjugate prior to measurement by spectrophotometer.

Notes:

• The protein concentration may also be determined colorimetrically; however, the dye quantification must be performed using a UV-Vis spectrophotometer.

• The typical recovery of dye-labeled proteins (14-190 kDA) purified by Pierce Zeba™ Desalting Spin Col-umns was found to be greater than 80%.

D P/ A780

εDye----------

A280 0.03 A780×( )–εProtein

------------------------------------------------÷=

Protein conc. (mg/ml)A280 0.03 A780–

Protein

------------------------------------------------ MWProtein

dilution factor=

100

90

80

70

60

50

40

30

20

10

0

No

rmal

ized

Ab

sorb

ance

Wavelength (nm)

250 300 350 400 450 500 550 600 650 700 750 800 850 900

Figure 3. The spectrum of a typical IgG labeled with IRDye 800CW is provided for reference.

LI-COR Biosciences

Page 6

VII. Handling of Labeled Conjugates

To improve shelf life and stability of labeled conjugates you may add sodium azide (0.01%) and bovine serum albumin (1 - 10 mg/mL). Higher amounts of sodium azide will cause degradation of the dye and should be avoided. Note that the BSA should only be added after the D/P ratio is determined, as BSA absorbs at 280 nm and will interfere with D/P ratio calculation. Do not add BSA or sodium azide to samples for

in vivo

imaging applications. IgG conjugates should be stored at 4°C and protected from light. They are stable at 4°C for up to six months, or they can be frozen in small aliquots for longer-term storage. For conjugated proteins other than IgGs, optimal storage conditions and buffers may vary. However, all dye conjugates should be protected from light. Avoid freeze-thaw cycles of conjugates, as this will greatly reduce performance.


For best results, read and follow the protocol carefully.

Notes and Tips

■

The protocol is optimized for a protein concentration of 1 mg/mL. Deviations from this amount will affect the D/P ratio.

■

The pH of the reaction is critical. The reaction should be run using phosphate buffer at pH 8.5.

■

It is important to note that the labeling reaction will continue even after the suggested 2-hour reaction time, if NHS ester is still present.

■

Under-labeling

: Different proteins and antibodies will react with the fluorophore at different rates. For this reason, the standard protocol provided here may not always produce optimal labeling. To label with a higher D/P ratio, try re-labeling the same protein sample, or perform a new reaction with fresh protein sample using either less protein or more of the reactive dye to increase the molar ratio of dye to protein in the reaction.

■

Over-labeling

: If a protein is too heavily labeled, it may not function well in the intended assay. Over-labeling can cause fluorophore quenching (which reduces desired signal), aggregation and nonspecific staining (which increases assay background). Over-labeling may also affect biological/biochemical activity, biodistri-bution, or clearance of conjugates

in vivo

. If over-labeling occurs, reduce the degree of labeling in subse-quent reactions by adding more protein or reducing the amount of reactive dye added. Either approach will decrease the molar ratio of dye to protein in the reaction.

■

Preparation of protein conjugates for other applications

: The optimal degree of labeling may vary for different applications. For

in vivo

imaging applications, the dye/protein ratio of the conjugate may affect biological or biochemical activity of the protein, signal-to-noise ratio, blood clearance, and biodistribution (for example, Schellenberger et al., 2004). The optimal degree of labeling for different proteins may vary widely, so we rec-ommend that you prepare several conjugates with different D/P ratios for evaluation in your desired applica-tion.

■

Labeling of proteins:

It is recommended that you perform a pilot labeling reaction using the conditions described in this protocol and evaluate the resulting D/P ratio. If necessary, the labeling conditions can then be altered to change the D/P ratio. As a general rule, lower MW proteins should be labeled with fewer dye molecules, while higher MW proteins can tolerate a higher degree of labeling. Please note that when calcu-lating the labeling ratio of the conjugate (section VI) you must use the correct extinction coefficient for the protein.

IRDye

®


Page 7

Troubleshooting


Protein is over-labeled (D/P ratio is too high).

Free dye present (i.e. the real amount of labeling may be less than it appears)

Process conjugates though a second spin column to remove free dye.

Reactive dye and/or protein con-centration in labeling reaction not optimal

Use less dye or more protein in the labeling reaction.

Temperature of labeling reac-tion too high

Perform reaction at 20 - 25 °C.

Labeling reaction carried out too long

Incubate reaction for 2 hours. Purify by spin column immediately.

Protein is under-labeled(D/P ratio is too low).

Reactive dye and/or protein con-centration in the labeling reac-tion not optimal

Re-label the conjugate to increase the D/P ratio.

Protein with inherently low labeling efficiency

Use more dye.

Reactive contaminant(s) or pre-servatives present in original protein solution

Dialyze or desalt unlabeled protein prior to labeling reaction.

NHS ester content too low Use a fresh vial of dye for labeling reaction. Do not allow dye solution to stand for more than a few minutes before use.

Protein concentration too low Concentrate protein before labeling reaction.

High background in Westerns.

Excessive free dye Process conjugates through a second spin column to remove free dye.

Over-labeling of protein Recheck D/P ratio and possibly repeat labeling reaction.

Poor blocking Try a different blocking buffer.

Try a different membrane.

LI-COR Biosciences

Page 8

IX. Reference

Schellenberger, E.A., R. Weissleder, and L. Josephson. 2004. Optimal modification of annexin V with fluorescent dyes.

Chembiochem.

5:271-274.

Low signal in Westerns. Protein over-labeled Over-labeling can cause self-quench-ing of the dye, leading to reduced sig-nal and higher background.

Protein under-labeled Re-label conjugate to increase D/P ratio.

Inappropriate blocker used Try a different blocking buffer. Primary antibody performance is highly depen-dent on choice of blocker.

High background in In-Cell Westerns.

Excessive free dye In-Cell Westerns are very sensitive to free dye; process conjugates though a second spin column to remove free dye.

Over-labeling D/P ratios higher than 2:1 will cause excess background in this assay. Label new antibody with a lower D/P ratio.

Distorted absorption spectrum.

Some distortion in 1X PBS is normal, especially at high D/P ratio.

Record UV/Vis spectrum in 1:1 Methanol: PBS.

Distortion observed when protein is over-labeled

Use less dye or more protein in label-ing reaction.


IRDye

® 800CW Protein Labeling Kit - High MW

Page 9

Limitation of Liability and Limited Use Label License

LI-COR IRDye® infrared dyes are offered for research purposes only and are not intended for human therapeutic or diagnostic use. The purchase of this product conveys to the buyer the non-transferable right to use the amount of product purchased and the components of the product in research conducted by the buyer (whether the buyer is a not-for-profit, academic or for-profit entity). The buyer shall not sell or otherwise transfer this product, its components, or materials made therefrom to any third party. Buyer shall not use this product or its components for commercial purposes. The term “commercial purposes” shall mean any activity by a party for consideration and may include, but is not limited to, use of the product or its components (i) in manufacturing, (ii) to provide a service, information or data, (iii) for therapeutic, diagnostic or prophylactic purposes, or (iv) for resale, whether or not such product or its components are resold for use in research. Buyer shall not determine the structure or otherwise reverse engineer this product. The use of this product by the buyer constitutes agreement with the terms of this limited use label license for LI-COR IRDye® infrared dyes. Inquiries regarding the licensing of one or more IRDye® infrared dyes should be submitted by e-mail to [email protected].

LI-COR DOES NOT PROVIDE RESEARCH ADVICE OR DETERMINE OR RECOMMEND ANY POTENTIAL USES FOR IRDYE® INFRARED DYES. LI-COR MAKES NO WARRANTIES OF ANY KIND, EITHER EXPRESS OR IMPLIED, AS TO ANY MATTER INCLUDING, BUT NOT LIMITED TO, WARRANTY OF FITNESS FOR PURPOSE, OR MERCHANTABILITY OR RESULTS OBTAINED FROM USE OF IRDYE® INFRARED DYES. IN NO EVENT SHALL LI-COR BE LIABLE FOR LOST PROFITS, CONSEQUENTIAL, EXEMPLARY, SPECIAL, DIRECT, INCIDENTAL, OR PUNITIVE DAMAGES, OR ATTORNEY FEES, EVEN IF LI-COR HAD BEEN ADVISED OF, KNEW OR SHOULD HAVE KNOWN, OF THE POSSIBILITIES THEREOF. NO EMPLOYEE, AGENT OR REPRESENTATIVE OF LI-COR HAS THE AUTHORITY TO BIND LI-COR TO ANY ORAL REPRESENTATION OR WARRANTY EXCEPT AS SPECIFICALLY SET FORTH HEREIN.





www.licor.com

LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey, Aerius, and IRDye are registered trademarks of LI-COR, Inc. Pierce is a registered trademark and Zeba is a trademark of Pierce Biotechnology Inc. The Odyssey Infrared Imager, Aerius Automated Infrared Imaging System, IRDye

®

800CW and IRDye

®

infrared dyes are covered by U.S. and foreign patents and patents pending.

®

Doc# 988-08616

®

IRDye 800CW Protein Labeling Kit - Low MW


®

IRDye


®


IRDye

®

800CW Protein Labeling Kit - Low MW

Page 1

Contents

Page


IX. Reference .........................................................................................................8

I. Introduction

The IRDye

®

800CW Protein Labeling Kit - Low MW is optimized to label proteins for use with the Odyssey

®

Infrared Imaging System, Aerius™ Automated Imaging System or other

in vivo


in vivo

imaging and other applications.

The kit is optimized for labeling 1 mg of protein with molecular weight 15 - 45 kDa. For proteins of higher molecular weight, use IRDye 800CW Protein Labeling Kit - High MW (P/N 928-38040). For small amounts of protein (100 µg) with molecular weight 14-200 kDa, use IRDye 800CW Protein Labeling Kit - Microscale (P/N 928-38044).

The IRDye 800CW dye bears an NHS ester reactive group that will couple to proteins and form a stable conjugate. Fluorescent conjugates labeled with IRDye 800CW display an absorption maximum of 774 nm and an emission maximum of 789 nm in 1X PBS (see Figure 1). These spectral characteristics match the 800 nm channel on the Odyssey and Aerius.


LI-COR Biosciences

Page 2

II. Kit Components


2

HPO

4


®


Note:

The minimum recommended protein molecular weight for these columns is 7 kDa. • Pierce Zeba™ Desalting Spin Column instructions • Protocol for IRDye 800CW Protein Labeling Kit - Low MW



1. Prepare 1.0 mg of protein in a phosphate buffer without sodium azide, at a concentration of 1 (± 0.1) mg/ml.

Notes:




• Unpurified proteins and protein solutions that contain BSA or other proteinaceous stabilizers will not label well and have not been characterized for use with this kit.


2

HPO

4


th



Note:


IRDye

®


Page 3



Notes:


• The optimal degree of labeling will vary for different applications. The effect of labeling on the biological activity of the protein will depend on factors including size and amino acid composition. Over-labeling may cause high background or self-quenching of the dye.


• For

in vivo


et al.

, 2004).


Note:


3. Mix the appropriate amount of dye with 1.0 mg of protein. React for 2 hours at 20°C, protecting the vial from light.

Note:

• Little difference in labeling efficiency was noted from 4-25 °C. However, the kit is optimized for reactions at 20 °C. If the labeling reaction is performed at temperatures less than 20°C, any remaining reactive dye must be removed from the sample immediately after the 2 hour incubation (see Section V). At 20-25 °C, there is little or no reactive dye remaining in the reaction mix after the 2 hour incubation.


Dye Volume = 233.2 MW

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

0 10 20 30 40 50

MW (kDa)

Dye

Vo

lum

e (µ

L)

5.1845

5.8340

6.6635

7.7730

9.3325

11.720

15.515

(µL)(kDA)

Dye VolMW


LI-COR Biosciences

Page 4



The Pierce Zeba™ Desalting Spin Columns are suitable for 0.5 - 2.0 mL of reaction volume. Other sizes are available from the manufacturer. The recommended molecular weights for the spin columns are > 7 kDa.


Once the free dye has been sufficiently removed, the dye to protein ratio of the conjugate can be deter-mined. Calculate the number of dye molecules per protein molecule (dye/protein or D/P ratio) by measur-ing absorbance with a UV-Vis spectrophotometer.

Important:



Note:




Note:



780


280



280

and A

780

).

IRDye

®


Page 5


In which:

■


■

ε

Dye

and

ε

Protein


■

ε

Dye

is 270,000 M

-1

cm

-1

in a 1:1 mixture of PBS:Methanol.

■

ε

Protein

can be determined spectrophotometrically or found in the literature.

Use of the correct extinction coefficient for your protein is essential for accurate determination of the D/P ratio.


In which:

■

MW

Protein


■


Notes:



D P/ A780

εDye----------

A280 0.03 A780×( )–εProtein

------------------------------------------------÷=


Protein

------------------------------------------------ MWProtein

dilution factor=

LI-COR Biosciences

Page 6


To improve shelf life and stability of labeled conjugates you may add sodium azide (0.01%) and bovine serum albumin (1 - 10 mg/mL). Higher amounts of sodium azide will cause degradation of the dye and should be avoided. BSA should only be added after the D/P ratio is determined, as BSA absorbs at 280 nm and will interfere with D/P ratio calculation. Do not add BSA or sodium azide to samples for

in vivo

imaging applications. Optimal storage conditions and buffers may vary depending on the protein. However, all dye conjugates should be protected from light. Avoid freeze-thaw cycles of conjugates, as this will greatly reduce performance.



Notes and Tips

■


■


IRDye 800CW Lactalbumin in 1:1 PBS:Methanol

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

250 350 450 550 650 750 850

W a v e le n g th (n m )

Figure 3. The spectrum of a typical protein labeled with IRDye 800CW is provided for reference.

250 350 450 550 650 750 850

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0

Ab

sorb

ance

(au

)

Wavelength (nm)

IRDye 800CW Lactalbumin in 1:1 PBS:Methanol

IRDye

®


Page 7

■

It is important to note that the labeling reaction can continue even after the suggested 2-hour reaction time, if NHS ester is still present.

■

Under-labeling:

Different proteins will react with the fluorophore at different rates. For this reason, the stan-dard protocol provided here may not always produce optimal labeling. To label with a higher D/P ratio, try re-labeling the same protein sample, or perform a new reaction with fresh protein sample using either less protein or more of the reactive dye to increase the molar ratio of dye to protein in the reaction.

■

Over-labeling:

If a protein is too heavily labeled, it may not function well in the intended assay. Over-labeling can cause fluorophore quenching (which reduces desired signal), aggregation and nonspecific staining (which increases assay background). Over-labeling may also affect biological/biochemical activity, biodistri-bution, or clearance of conjugates

in vivo


■

Preparation of protein conjugates for other applications:

The optimal degree of labeling may vary for different applications. For

in vivo


■



Troubleshooting







Temperature of labeling reaction too high




LI-COR Biosciences

Page 8

IX. Reference


Chembiochem.

5:271-274.





Use more dye.






IRDye

®


Page 9



LI-COR DOES NOT PROVIDE RESEARCH ADVICE OR DETERMINE OR RECOMMEND ANY POTENTIAL USES FOR IRDYE® INFRARED DYES. LI-COR MAKES NO WARRANTIES OF ANY KIND, EITHER EXPRESS OR IMPLIED, AS TO ANY MATTER INCLUDING, BUT NOT LIMITED TO, WARRANTY OF FITNESS FOR PURPOSE, OR MERCHANTABILITY OR RESULTS OBTAINED FROM USE OF IRDYE® INFRARED DYES. IN NO EVENT SHALL LI-COR BE LIABLE FOR LOST PROFITS, CONSE-QUENTIAL, EXEMPLARY, SPECIAL, DIRECT, INCIDENTAL, OR PUNITIVE DAMAGES, OR ATTORNEY FEES, EVEN IF LI-COR HAD BEEN ADVISED OF, KNEW OR SHOULD HAVE KNOWN, OF THE POSSIBILITIES THEREOF. NO EMPLOYEE, AGENT OR REPRESENTATIVE OF LI-COR HAS THE AUTHORITY TO BIND LI-COR TO ANY ORAL REPRESENTATION OR WAR-RANTY EXCEPT AS SPECIFICALLY SET FORTH HEREIN.





www.licor.com


®

800CW and IRDye

®


®

Doc# 988-08617

®

IRDye 800CW Protein Labeling Kit - Microscale


®

IRDye


®


IRDye

®

800CW Protein Labeling Kit - Microscale

Page 1

Contents

Page


IX. Reference .........................................................................................................8

I. Introduction

The IRDye

®

800CW Protein Labeling Kit - Microscale is optimized to label proteins for use with the Odyssey

®

Infrared Imaging System, Aerius™ Automated Imaging System or other

in vivo


in vivo

imaging, and other applications.

The kit is optimized for labeling 100 µg of protein with molecular weight 14 - 200 kDa. For larger amounts of protein (1 mg) with molecular weight 14 - 45 kDa, use IRDye 800CW Protein Labeling Kit - Low MW (P/N 928-38042). For larger amounts of protein with molecular weight 45 - 200 kDA, use IRDye 800CW Protein Labeling Kit - High MW (P/N 928-38040).

The IRDye 800CW dye bears an NHS ester reactive group that will couple to proteins and form a stable conjugate. Fluorescent conjugates labeled with IRDye 800CW display an absorption maximum of 774 nm and an emission maximum of 789 nm in 1X PBS. These spectral characteristics match the 800 nm channel on the Odyssey and Aerius.


LI-COR Biosciences

Page 2

II. Kit Components


2

HPO

4


®


Note:

The minimum recommended protein molecular weight for these columns is 7 kDa. • Pierce Zeba™ Desalting Spin Column instructions • Protocol for IRDye 800CW Protein Labeling Kit - Microscale



1. Prepare 100 µg of protein in a phosphate buffer without sodium azide, at a concentration of 1 (± 0.1) mg/mL.

Notes:




• Unpurified antibodies (such as ascites fluid and crude serum), cell lysates, and proteins that contain BSA or other proteinaceous stabilizers will not label well and have not been characterized for use with this kit.


2

HPO

4


th



Note:


IRDye

®


Page 3



Notes:


• The optimal degree of labeling will vary for different applications, and the effect of labeling on the biologi-cal activity of the protein will depend on factors including size and amino acid composition. Over-labeling may cause high background or self-quenching of the dye.


• A D/P ratio of 1:1 - 2:1 for an IgG antibody is suitable for both Western and In-Cell Western applications. Higher D/P ratios (3:1 - 4:1) for an IgG antibody may still be usable for Western blot detection, but may exhibit increased background and therefore not perform optimally for other applications.

• For

in vivo


et al

., 2004).


Note:


3. Mix the appropriate amount of dye with 100 µg of protein. React for 2 hours at 20°C, protecting the vial from light.

Note:

• Little difference in labeling efficiency was noted from 4-25°C. However, the kit is optimized for reactions at 20°C. If the labeling reaction is performed at temperatures less than 20°C, any remaining reactive dye must be removed from the sample immediately after the 2 hour incubation (see Section V). At 20-25°C, there is little or no reactive dye remaining in the reaction mix after 2 hour incubation.


Dye Volume = 116.6 MW

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

0 50 100 150 200 250

MW (kDa)

Dye

Vo

lum

e (µ

L)

0.58200

0.73160

1.17100

1.4680

2.3350

3.8930

7.7715

(µL)(kDA)

Dye VolMW


LI-COR Biosciences

Page 4



The Pierce Zeba™ Desalting Spin Columns are suitable for 30 - 130 µL of reaction volume. Other sizes are available from the manufacturer. The recommended molecular weights for the spin columns are > 7 kDa.


Once the free dye has been sufficiently removed, the dye to protein ratio of the conjugate can be determined. Calculate the number of dye molecules per protein molecule (dye/protein or D/P ratio) by measuring absorbance with a UV-Vis spectrophotometer.

Important:



Note:




Note:



780


280



280

and A

780

).

IRDye

®


Page 5


In which:

■


■

ε

Dye

and

ε

Protein


■

ε

Dye

is 270,000 M

-1

cm

-1

and

ε

Protein

is 203,000 M

-1

cm

-1

(for a typical IgG) in a 1:1 mixture of PBS:Methanol.

Proteins other than IgG may have very different molar extinction coefficients; use of the correct extinction coefficient for your protein is essential for accurate determination of the D/P ratio.


In which:

■

MW

Protein


■


Notes:



D P/ A780

εDye----------

A280 0.03 A780×( )–εProtein

------------------------------------------------÷=


Protein

------------------------------------------------ MWProtein

dilution factor=

100

90

80

70

60

50

40

30

20

10

0

No

rmal

ized

Ab

sorb

ance

Wavelength (nm)

250 300 350 400 450 500 550 600 650 700 750 800 850 900

Figure 3. The spectrum of a typical IgG labeled with IRDye 800CW is provided for reference.

LI-COR Biosciences

Page 6


To improve shelf life and stability of labeled conjugates you may add sodium azide (0.01%) and bovine serum albumin (1 - 10 mg/mL). Higher amounts of sodium azide will cause degradation of the dye and should be avoided. BSA should be added only after the D/P ratio is determined, as BSA absorbs at 280 nm and will interfere with D/P ratio calculation. Do not add BSA or sodium azide to samples for

in vivo

imaging applications. IgG conjugates should be stored at 4 °C and protected from light. They are stable at 4 °C for up to six months, or they can be frozen in small aliquots for longer-term storage. For conjugated proteins other than IgGs, optimal storage conditions and buffers may vary. However, all dye conjugates should be protected from light. Avoid freeze-thaw cycles of conjugates, as this will greatly reduce performance.



Notes and Tips

■


■


■

It is important to note that the labeling reaction can continue even after the suggested 2-hour reaction time, if NHS ester is still present.

■

Under-labeling:

Different proteins and antibodies will react with the fluorophore at different rates. For this reason, the standard protocol provided here may not always produce optimal labeling. To label with a higher D/P ratio, try re-labeling the same protein sample, or perform a new reaction with fresh protein sample using either less protein or more of the reactive dye to increase the molar ratio of dye to protein in the reaction.

■

Over-labeling:

If a protein is too heavily labeled, it may not function well in the intended assay. Over-labeling can cause fluorophore quenching (which reduces desired signal), aggregation and nonspecific staining (which increases assay background). Over-labeling may also affect biological/biochemical activity, biodistri-bution, or clearance of conjugates

in vivo


■

Preparation of protein conjugates for other applications:

The optimal degree of labeling may vary for different applications. For

in vivo


■



IRDye

®


Page 7

Troubleshooting







Temperature of labeling reac-tion too high








Use more dye.





High background in Westerns.

Excessive free dye Process conjugates through a second spin column to remove free dye.

Over-labeling of protein Recheck D/P ratio and possibly repeat labeling reaction.

Poor blocking Try a different blocking buffer.

Try a different membrane.

LI-COR Biosciences

Page 8

IX. Reference


Chembiochem.

5:271-274.

Low signal in Westerns. Protein over-labeled Over-labeling can cause self-quench-ing of the dye, leading to reduced sig-nal and higher background.

Protein under-labeled Re-label conjugate to increase D/P ratio.

Inappropriate blocker used Try a different blocking buffer. Primary antibody performance is highly depen-dent on choice of blocker.

High background in In-Cell Westerns.

Excessive free dye In-Cell Westerns are very sensitive to free dye; process conjugates though a second spin column to remove free dye.

Over-labeling D/P ratios higher than 2:1 will cause excess background in this assay. Label new antibody with a lower D/P ratio.

Distorted absorption spectrum.

Some distortion in 1X PBS is normal, especially at high D/P ratio.

Record UV/Vis spectrum in 1:1 Methanol: PBS.

Distortion observed when protein is over-labeled

Use less dye or more protein in label-ing reaction.


IRDye

® 800CW Protein Labeling Kit - Microscale

Page 9



LI-COR DOES NOT PROVIDE RESEARCH ADVICE OR DETERMINE OR RECOMMEND ANY POTENTIAL USES FOR IRDYE® INFRARED DYES. LI-COR MAKES NO WARRANTIES OF ANY KIND, EITHER EXPRESS OR IMPLIED, AS TO ANY MATTER INCLUDING, BUT NOT LIMITED TO, WARRANTY OF FITNESS FOR PURPOSE, OR MERCHANTABILITY OR RESULTS OBTAINED FROM USE OF IRDYE® INFRARED DYES. IN NO EVENT SHALL LI-COR BE LIABLE FOR LOST PROFITS, CONSEQUENTIAL, EXEMPLARY, SPECIAL, DIRECT, INCIDENTAL, OR PUNITIVE DAMAGES, OR ATTORNEY FEES, EVEN IF LI-COR HAD BEEN ADVISED OF, KNEW OR SHOULD HAVE KNOWN, OF THE POSSIBILITIES THEREOF. NO EMPLOYEE, AGENT OR REPRESENTATIVE OF LI-COR HAS THE AUTHORITY TO BIND LI-COR TO ANY ORAL REPRESENTATION OR WARRANTY EXCEPT AS SPECIFICALLY SET FORTH HEREIN.





www.licor.com


®

800CW and IRDye

®


®

Doc# 988-08618

®

In-Cell Western AssayKits I and II

Published November, 2006. The most recent version of this protocol is posted at http://biosupport.licor.com/protocols.jsp

™

Odyssey and Aerius

Infrared Imaging Systems

®

Part Numbers: 926-31070 and 926-31072

Storage: 4 °C

®

In-Cell Western

™

Assay Kit I and Kit II Pack Insert

Page 1

The In-Cell Western Kit provides detection reagents for cell-based In-Cell Western™ Assays. The kit includes blocking buffer, fluorescently labeled IRDye

®

800CW secondary antibody for detection of a spe-cific protein target in the 800 nm channel, and two fluorescent cell stains that are used in combination in the 700 nm channel to normalize for well-to-well variations in cell number. This approach allows the target of interest to be detected accurately and cost-effectively.

If your application requires you to discriminate two protein targets with two different primary antibodies, this can be accomplished using secondary antibodies labeled with IRDye 680 and IRDye 800CW in a mul-tiplex assay. Secondary antibodies for multiplex detection can be purchased at www.licor.com.

Using DRAQ5™ and Sapphire700™ for Cell Number Normalization

The cell stains included in this kit are designed to be used in combination, to provide accurate normaliza-tion over a broad range of cell densities. DRAQ5™ is a cell permeable DNA-interactive agent that can be used for stoichiometric staining of DNA in live or fixed cells. DRAQ5 is part of this kit, but is otherwise sold separately by Biostatus Limited (visit http://www.biostatus.com/product/draq5/). When serial dilutions of A431 human epithelial carcinoma cells are plated in 96-well plates, DRAQ5 demonstrates linearity of fluo-rescent signal for lower cell densities, up to ~50,000 cells/well (Figure 1A).

Sapphire700™ is a non-specific cell stain that accumulates in both the nucleus and cytoplasm of fixed or dead cells, but

not

live cells. When used to stain serial dilutions of A431 cells in 96-well plates, Sapphire700 displays linearity of fluorescent signal for higher cell densities, from ~50,000 to ~250,000 cells/well (Figure 1B). Simultaneous staining of cells with both DRAQ5 and Sapphire700 expands the linear range, allowing more accurate normalization of cell number across both low and high cell densities (Figure 1A and B). Sapphire700 can be purchased separately from LI-COR (Cat# 928-40022)

In Cell Western assays commonly use primary and secondary antibodies for normalization in the 700 nm channel. For example, if phospho-ERK is the target of interest, an antibody against total ERK (or against a housekeeping protein) can be used to normalize for variations in cell number. Staining with DRAQ5 and Sapphire700 eliminates the need for this additional primary and secondary antibody, and yields the same quantitative measurement of ERK phosphorylation (Figure 2).

A) B)

DRAQ5TM

+Sapphire700 Cell Linearity

0

20000

40000

60000

80000

100 1000 10000 100000 1000000Cell Number (A431)

70

0C

h.

Re

lati

ve

Inte

ns

ity DRAQ5 R(2)=0.9688

Sapphire700 R(2)=0.6785DRAQ5+Sapphire700 R(2)=0.9959

DRAQ5TM+Sapphire700 Cell Linearity

0

10000

20000

30000

40000

50000

60000

0 50,000 100,000 150,000 200,000 250,000 300,000

Cell Number (A431)

70

0C

h.

Re

lati

ve

Inte

ns

ity DRAQ5 R(2)=0.7814

Sapphire700 R(2)=0.9158DRAQ5+Sapphire700 R(2)=0.9821

Figure 1. DRAQ5 and Sapphire700 as normalizing agents for In-Cell Western assays. Dilutions of A431 cells were plated on clear, flat bottom 96-well plates, then fixed and permeabilized. Cells were stained with DRAQ5 alone, Sapphire700 alone, or both stains combined. A) Two-fold dilutions of cells, over a wide range of cell densities (0 - 200,000 cells/well). B) Closer examination of linearity of signal over the range of 25,000 - 250,000 cells/well, in dilution increments of 25,000 cells.

LI-COR Biosciences

Page 2

In-Cell Western Kit: Protocol for Use

Kit Components (store kit at 4°C)

• IRDye 800CW-labeled secondary antibody, 0.5 mg (lyophilized)• Odyssey Blocking Buffer, 4 x 500 ml (LI-COR, Cat# 927-40000)• DRAQ5, 100 µl • Sapphire700, 100 µl (LI-COR, Cat# 928-40022)

Additional Reagents (required but not included)

• Primary antibody• 1X PBS wash buffer (LI-COR, Cat# 928-40018, 10X PBS)• Tissue culture reagents (serum D-MEM, trypsin, etc.)• Clear or black 96-well microplate (see

V. Hints and Tips for In-Cell Western Assays

)• 37% formaldehyde• 20% Tween

®

-20• 10% Triton

®

X-100

19 .9

18.3

0

6

12

18

24pERK/ERKpERK/DRAQ5+Sapphire

0 .8 0.8

0

0 .3

0 .6

0 .9

1 .2pERK/ERKpERK/DRAQ5+Sapphire

Fold-activation of ERKBackground

800

Ch

Inte

gra

ted

Inte

nsi

ty

Rel

. Fllu

ore

scen

ce In

ten

sity

TotalERK

Overlay 700 nm (normalization) 800 nm (pERK)

DRAQ5/Sapphire700

TotalERK

DRAQ5/Sapphire700

TotalERK

DRAQ5/Sapphire700

Figure 2. Comparison of normalization methods. ERK activation was induced in A431 cells by stimulation with epidermal growth factor (EGF). Phospho-ERK was detected in the 800 nm channel with anti-phospho-ERK primary antibody and IRDye 800CW secondary antibody. Normalization was performed in two ways: anti-total-ERK primary antibody and IRDye 680 secondary antibody; or DRAQ5 and Sapphire700 staining. The EGF-induced ERK activation measured by the two methods was indistinguishable.

In-Cell Western

™


Page 3

I. Reconstitution of Antibody

II. Cell Preparation and Fixation

1. Protect from light. Store IRDye 800CW secondary antibody at 4 °C prior to reconstitution.

2. Reconstitute contents of antibody vial with 0.5 ml sterile distilled water. Mix gently by inverting, and allow to rehydrate for at least 30 minutes before use. Centrifuge product if solution is not completely clear after standing at room temperature.

3. Dilute only immediately prior to use. Reconstituted antibody is stable for 3 months at 4 °C when stored undiluted as directed. For extended storage, aliquot and freeze at -20 °C or below; avoid repeated freeze-thaw cycles.

1. Treat cells as desired with drug, stimulant, etc. Detailed In-Cell Western protocols for certain cell lines and target proteins may be downloaded at http://biosupport.licor.com/protocols.jsp.

2. Remove media manually or by aspiration. Immediately fix cells with

Fixing Solution

(3.7% formaldehyde in 1X PBS) for 20 minutes at room temperature (RT).

a. Prepare fresh

Fixing Solution

as follows:



b. Using a multi-channel pipettor, add 150 µl of fresh, room temperature

Fixing Solution

to each well. Add the

Fixing Solution


c. Allow incubation on the bench top for 20 minutes at RT with no shaking.

Note:

• If optimal fixation conditions for immunofluorescent staining of your cell line and/or target protein are already known, these conditions may be more appropriate than the fixation protocol described here and would be an excellent starting point for In-Cell Western assay development. Most fixatives and fixation pro-tocols for immunofluorescent staining may be adapted to the In-Cell Western format.

LI-COR Biosciences

Page 4

III. Cell Staining

3. To permeabilize, wash five times with 1X PBS containing 0.1% Triton X-100 for 5 minutes per wash.

a. Prepare


as follows:


1X PBS + 0.1% Triton X-100 500 ml

b. Remove

Fixing Solution


c. Using a multi-channel pipettor, add 200 µl of room temperature


to each well. Make sure to carefully add the solution down the sides of the wells to avoid detaching the cells.

d. Allow wash to shake on a rotator for 5 minutes.

e. Repeat washing steps 4 more times, removing wash manually each time. Do not allow cells/wells to become dry during washing. Immediately add the next wash after each manual disposal.

Note:

• If an alternative permeabilization method (for example, ice-cold methanol) is known to work well for immunofluorescent staining of your protein target, you may prefer to use that permeabilization method rather than the Triton method described here.

1. Using a multi-channel pipettor, block cells by adding 150 µl of Odyssey Blocking Buffer to each well.


Hints and Tips for Blocking:

• No single blocking reagent will be optimal for every antigen-antibody pair. Some primary antibodies may exhibit greatly reduced signal or nonspecific binding in different blocking solutions. If you have difficulty detecting your target protein, changing the blocking solution may dramatically improve performance. If you have used the primary antibody successfully for immunofluorescent staining, consider trying the same blocking buffer for In-Cell Western detection.

• Odyssey Blocking Buffer often yields higher and more consistent sensitivity and performance than other blockers. Nonfat dry milk or casein dissolved in PBS, or commercial blocking buffers, can also be used for blocking and antibody dilution. When using anti-goat antibodies, milk-based reagents may be contami-nated with endogenous IgG, biotin, or phospho-epitopes that can interfere with detection.

2. Allow blocking for 1.5 hours at room temperature with moderate shaking on a rotator.

In-Cell Western

™


Page 5

3. Dilute desired primary antibody in Odyssey Blocking Buffer or other appropriate blocker. As a general guideline, 1:50 to 1:200 dilutions are recommended depending on the primary antibody. If the antibody supplier provides dilution guidelines for immunofluorescent staining, start with that recommended range.

Note:

• If using DRAQ5 and Sapphire700 for normalization, only one primary antibody will be used. Alternatively, you may choose to normalize with a second primary antibody in your assay. The second primary antibody MUST be from a different host, and an appropriate IRDye 680 secondary antibody (not provided in the kit) will be required.

a. It is important to include control wells that DO NOT contain primary antibody. These wells will be treated with secondary antibody only, and should be used to correct for background staining in the data analysis.

b. Remove blocking buffer from step 2.

c. Add 50 µl of Odyssey Blocking Buffer to the control wells and 50 µl of the desired primary antibody in Odyssey Blocking Buffer to the rest of the wells.

4. Incubate with primary antibody for 2.5 hours at room temperature or overnight at 4 °C with gentle shaking.


a. Prepare


as follows:





. Make sure to carefully add the solution down the sides of the wells to avoid detaching the cells from the well bottom.

c. Allow wash to shake on a rotator for 5 minutes.


6. Dilute the fluorescently labeled secondary antibody in Odyssey Blocking Buffer or other appropriate blocker. The recommended dilution range is 1:200 to 1:1,200, with a suggested starting dilution of 1:800. The optimal dilution for your assay should be determined empirically. To lower background, add Tween-20 at a final concentration of 0.2% to the diluted antibody.

Avoid prolonged exposure of the antibody vials to light

.

a. Secondary antibody staining and normalization staining are carried out simultaneously. To stain for normalization, add DRAQ5 and Sapphire700 to the diluted secondary antibody solution and apply this mixture to the cells. Suggested dilutions for normalization stains: DRAQ5 - 1:2000; Sapphire700 - 1:1000.

b. For control wells (used to calculate background), do not add DRAQ5 and Sapphire700. Add only diluted secondary antibody to these wells.

LI-COR Biosciences

Page 6

IV. Imaging

V. Hints and Tips for In-Cell Western Assays


• Establish the specificity of your primary antibody by Odyssey Western blotting of similar lysates, and/or by immunofluorescent microscopy. To achieve the most consistent results, use the same blocking buffer for vali-dation experiments and In Cell Western assays. If significant non-specific banding is detected on a Western blot, choose alternative primary antibodies. Non-specific binding of primaries will make it difficult to accu-rately interpret In-Cell Western assay results.



7. Add 50 µl of secondary antibody solution

without

DRAQ5 and Sapphire700 into each of the control wells and 50 µl of secondary antibody solution

with

DRAQ5 and Sapphire700 into rest of wells. Incubate for 1 hour at room temperature with gentle shaking.

Protect plate from light during incubation

.

8. Wash the plate five times with 1X PBS + 0.1% Tween-20 for 5 minutes at room temperature with gentle shaking, using a generous amount of buffer.



. Make sure to carefully add the solution down the sides of the wells to avoid detaching the cells from the well bottom.

b. Allow wash to shake on a rotator for 5 minutes.

c. Repeat washing steps 4 more times.



2. Before plate scanning, clean the bottom plate surface and the Odyssey Imager scanning bed with moist lint-free paper.

3. Scan the plate with detection in both the 700 and 800 channels, using the Odyssey or Aerius imagers (700 nm detection for normalization stains, and 800 nm detection for IRDye 800CW antibody). Generally, a scan resolution of 169 µm (Odyssey) or 200 µm (Aerius) is appropriate. An initial intensity setting of 5 is suggested for both 700 and 800 nm channels. Focus offset position is critical to proper plate imaging; for more information about choosing the correct focus offset, see Section V.

4. Proceed with data analysis.

• In-Cell Western assays require sterile plates for tissue culture growth. The following plates and focus offset set-tings are recommended as a starting point. Please be aware that the manufacturers' specifications for culture plates are subject to change.

* For use with suspension cells. For more information, please refer to the suspension cell protocol available in the Application Protocols Manual or on the LI-COR web site (http://biosupport.licor.com/protocols.jsp).

• The Odyssey and Aerius Imagers require that microplates have a maximum 4.0 mm distance from the scan-ning surface to the target detection area of the plate. Optimal signal will be achieved when the focus offset position is set as accurately as possible, and the best offset for your experiments may need to be determined empirically (see below).

• Determining the best focus offset is especially important when using plates other than those listed in the table above. Find the optimal focus offset by scanning a plate containing experimental and control samples at 0.5, 1.0, 2.0, 3.0, and 4.0 mm focus offsets. Use the same intensity settings for each scan. After reviewing the collected scans, use the focus offset with the highest signal-to-noise for your experiments. Focus offset can be further fine-tuned in 0.1-0.5 mm increments if desired, and this may result in additional improvement in signal strength.


• Intensity settings for both 700 and 800 nm channels should generally be set to 5 for initial scanning. If your image signal is saturated or too high, re-scan using a lower intensity setting (i.e. 2). If your image signal is too low, re-scan using a higher intensity setting (i.e. 7).





www.licor.com

LI-COR is an ISO 9001 registered company. © 2006 LI-COR Inc. LI-COR, Odyssey, Aerius, In-Cell Western Assay, Sapphire700 and IRDye are trade-marks or registered trademarks of LI-COR, inc. Tween is a registered trademark of ICI Americas, Inc. Triton X-100 is a registered trademark of Union Carbide Chemicals and Plastics Corporation. DRAQ5 is a trademark of Biostatus Limited. Nunc is a trademark of Nunc A/S Corporation. Falcon is a trademark of Becton Dickinson and Company. The Odyssey Infrared Imager is covered by U.S. patents, foreign equivalents, and patents pending.

Well Number Well Bottom Manufacturer Part Number Offset - Odyssey Offset - Aerius

96 Flat Nunc™-Nalgene 161093, 165305 3.0 mm 3.0 mm

96 Flat BD Falcon™ 353075, 353948 3.0 mm 3.0 mm

96 Round* BD Falcon 353077 3.0 mm

not recommended

96 Round* Nunc-Nalgene 163320

not recommended

3.9 mm

384 Flat Nunc-Nalgene 164688, 164730 3.0 mm 3.0 mm

384 Flat BD Falcon 353961, 353962 3.0 mm 3.0 mm

®

Doc# 988-08911

Documents

Table of Contents - Michigan State Universitycore.phmtox.msu.edu/Scheduling/ItemDocs/20/Odyssey... · LI-COR Biosciences Doc# 988-09288 Page 2 II. Western Detection Methods Nitrocellulose