ODYSSEY® Fc

IMAGING SYSTEM

QUANTITATIVE IR WESTERN BLOTS

INFRARED MULTIPLEX DETECTION

WIDE LINEAR DYNAMIC RANGE

CHEMILUMINESCENT DETECTION

HIGH SENSITIVITY

CLEAR DATA

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For ten years, the LI-COR® Biosciences’ Odyssey® Infrared

Imaging System has set the standard for quantitative Western

blot analysis. The Odyssey Fc Imager is the first CCD imager that

offers excellent infrared (IR) fluorescent and chemiluminescent

imaging performance in one system. The Odyssey Fc optical

system combines solid-state laser diodes at 685 and 785

nm with a low-noise, CCD camera for detection of IR and

chemiluminescent signals.

At the visible wavelengths used by most fluorescent imagers,

autofluorescence and light scatter produce high membrane

backgrounds. This limits sensitivity and makes it difficult to detect

low-abundance proteins at endogenous levels. IR fluorescence

detection allows you to quantitate proteins across a wide linear

dynamic range, because autofluorescence and light scatter are

greatly reduced. The result? The cleanest background, highest

signal-to-noise ratios, and best detection sensitivity available

with a fluorescent system.

Advantages of Infrared DetectionPlus Chemiluminescence

ODYSSEY® Fc

IMAGING SYSTEM

Quantitative IR Western Blots Direct infrared (IR) fluorescence detection

Infrared Multiplex Detection Detect multiple targets with two IR colors

Wide Linear Dynamic Range Unprecedented 4 million-fold (>6 log) range

Chemiluminescent Detection No darkroom or film needed

High Sensitivity Similar to industry-leading Odyssey®

Infrared Imaging System

Clear Data Image both weak and strong signals

Figure 1. Purified transferrin (60 ng/well) was spiked into serial dilutions of NIH/3T3 cell lysate (10 μg – 156 ng) and electrophoresed. The blot was probed with rabbit anti-transferrin and mouse anti-actin primary anti-bodies, followed by goat anti-rabbit IRDye® 680LT (in Red) and goat anti-mouse IRDye 800CW (in Green) secondary antibodies. Acquisition time was 2 min for each channel.

Multiplex Infrared Fluorescent Detection

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Chemiluminescence detection is simplified with the Odyssey Fc

Imaging System. Film and darkroom steps are eliminated, along

with the hazardous waste and excessive water usage of film

development.

Applications supported by the Odyssey Fc Imaging System

include Western blots (quantitative with IR fluorescence;

qualitative with chemiluminescence), protein and DNA gel

documentation, EMSA (gel shift assays), and In-Gel Westerns.

In addition, IR fluorescence can be used to visualize molecular

weight markers in the 700 nm channel on chemiluminescent

blots, eliminating the need for expensive HRP-conjugated

markers.

The Odyssey Fc Imaging System combines high sensitivity

with an exceptionally wide linear dynamic range. Now, both

low-abundance and high-abundance targets can be easily

detected and quantified on the same blot. LI-COR Biosciences’

Figure 2. Serial dilutions of purified human transferrin (10 ng – 1.2 pg) were used to assess Western blot sensitivity. The images represent four independent experiments. IR fluorescent blots were imaged with the Odyssey or Odyssey Fc Systems. Odyssey Fc sensitivity is comparable to the original Odyssey Imager. Chemiluminescent blots were incubated with ECL™ Plus substrate (GE Healthcare), and imaged with Odyssey Fc or exposed to film. Odyssey Fc matches or exceeds the sensitivity of film.

Comparison of IR Fluorescent and Chemiluminescent Detection

Odyssey (fluorescence)

Odyssey Fc (fluorescence)

Odyssey Fc (chemiluminescence)

Film (chemiluminescence)

FieldBrite™ XT technology acquires images over the full dynamic

range every time, with minimal user adjustments. With the

proprietary technologies of the Odyssey Fc Imaging System,

a single instrument can perform quantitative IR fluorescent

detection PLUS qualitative chemiluminescent detection.

Publication-quality images are easy to produce with either

detection method. (Because chemiluminescence is a time-

dependent enzymatic reaction, its quantitative range is limited. IR

fluorescence is superior for quantification.)

The Odyssey Fc Imaging System includes LI-COR Image Studio

Software for fast, easy image acquisition and analysis. At the

push of a button, targets of interest can be imaged over more

than six logs of linear dynamic range in every acquisition.

LI-COR® Biosciences’ proprietary FieldBrite XT technology

acquires images without saturated pixels on the first attempt,

with minimal user adjustments. With this new advance in CCD

imaging, high performance is achieved without supercooling

of the camera. A patented filtering technology guarantees low

total system noise for acquisition times up to one hour. As the

acquisition progresses, signal-to-noise ratio is optimized to

provide a superior image.

The Odyssey® Fc System provides 22-bit images, which deliver

more than six logs of linear dynamic range. This allows accurate

capture of both strong and weak signals on the same image.

FieldBrite™ XT Technology

FieldBrite XT offers:

n No need to supercool camera to reduce background noise

n One button acquisition for optimal image, with little or no

user intervention

n Even imaging over entire field of view

n Increased image acquisition time results in improved

signal to noise

Figure 4. ERK2 was detected in serial dilutions of A431 cell lysate (15 – 1.8 μg) using IRDye® 680 secondary antibody (LI-COR P/N 926-32220). The blot was imaged with the Odyssey Fc (A). Serial dilutions of transferrin (40 ng – 4.5 pg) were detected with IRDye 800CW secondary antibody (LI-COR P/N 926-32211). The blot was imaged with Odyssey Fc (B). For both targets. Odyssey Fc detec-tion showed no saturation.

Figure 5. IRDye® 800CW Streptavidin conjugate (10 μg – 1.2 pg) was sep-arated on a 10% Bis-Tris gel and transferred to PVDF membrane. Three replicate membranes were generated and imaged (10 min acquisition). Dynamic range of detection was 5.5 logs.

A

ERK2

B

Transferrin

μg IRDye 800CW Conjugate

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ence

Inte

nsity

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1010

109

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106

105

0.0001 0.001 0.01 0.1 1 10

The Odyssey Fc Imaging System is able to capture and quantify

the broad, linear dynamic range available with IR fluorescence.

This accuracy and linearity allows you to feel more confident

of the differences you detect in protein levels. IR blots can be

archived and imaged again months later, if needed.

In addition, the Odyssey Fc system can image chemiluminescent

signals. The dynamic enzymatic nature of chemiluminescence

allows you to capture only a “snapshot” of the enzymatic

reaction, and is highly dependent on timing and exposure.

This limits linear dynamic range and offers qualitative or semi-

quantitative results.

Importance of Linear Dynamic Range

Linear dynamic range – the region in which signal is directly

linearly proportional to the amount of target – is critical for

quantification. This value is often expressed in units of logs, or

10-fold ranges.

The Odyssey Fc System provides an unusually wide dynamic range for IR fluorescence – even wider than the original Odyssey Imager

(see Figure 6). Very high protein concentrations can be detected in a single image without signal saturation.

Accurate Quantification

n Detect changes in low-intensity bands (i.e., low-abundance

proteins)

n No need for multiple exposures

n Quantify weak and strong signals with a single image

n No saturation – more than 6 logs of linear dynamic range

Figure 6. Linear dynamic ranges of common detection methods

Chemiluminescence typically has a linear dynamic range of 1-2

logs1. With IR fluorescence, that range is much wider. The original

Odyssey Infrared Imaging System has a dynamic range of >4

logs. With FieldBrite XT technology, the Odyssey Fc System

provides an exceptional dynamic range of >6 logs (about 4

million-fold) to capture both low-abundance and high-abundance

targets in a single image.

The accuracy and linearity of the Odyssey Fc System ensure

confidence in quantification.

Wide Linear Dynamic Range of Fluorescent Detection

Chemiluminescenceand film

Odyssey® Infrared Imaging System

Odyssey® FcImaging System

Linear dynamic range 1-2 logs >4 logs >6 logs

Fold-change in signal intensity 10-100 >10,000 >1,000,000

Comparsion of values for detection method

~25 mL ~25 L~1.5 Olympic-size swimming pools

Applications

Western Blotting

IR fluorescent detection is a direct, nonenzymatic approach to

Western blotting that uses secondary antibodies labeled with

IRDye® infrared dyes. This method improves quantitative accuracy

and reproducibility, and facilitates multiplexing. The stable

fluorescent signal can be detected immediately, or after extended

storage.

LI-COR Biosciences provides protocols, application notes, helpful

tips and references to help you produce high-quality Western

blotting results. For more information, visit biosupport.licor.com.

Figure 7. The Odyssey Fc System can acquire images in both fluorescent and chemiluminescent modes. IR Fluorescence: Lasers are used to excite 700 nm and 800 nm IRDye fluorophores for 2 channel detection. Fluorescent emission is then detected to generate an image of the sample. At these IR wavelengths, mem-brane autofluorescence is very low. This method directly detects the fluorescently labeled conjugates. Because the fluorescent signal is very stable, samples can be archived and re-imaged. Chemiluminescence: Luminol substrate is applied to the sample, and is oxidized by horseradish peroxidase (HRP) enzyme in a light-generating reaction. This method is indirect, because it relies on the enzymatic reaction to report the location of the HRP conjugate. Chemiluminescent signals are dynamic and time-dependent. Light emission is initially strong, but diminishes over time.

IR Fluorescence

FieldBrite™ XTTechnology

FieldBrite™ XTTechnology

Fluorescence Light Generation

Laser Substrate

IRDye®

400-500nm

HRP

Chemiluminescence

HRP

Odyssey® Fc Detection Channels

Molecular weight markers can be visualized at 700 nm on chemiluminescent Western blotsFigure 8. NIH/3T3 cell lysates were separated and transferred to a mem-brane. Odyssey Protein MW Markers (P/N 928-40001, 928-40000) (which are labeled with 700 nm IR fluorescent dye) were included in the left lane. Akt was then detected, using appropriate antibodies and ECL Plus (GE Healthcare) chemiluminescent substrate.

Akt signal was acquired with the Odyssey Fc System in the chemilumines-cence channel (white bands) and 700 nm fluorescence channel (MW marker; red bands). The two signals were then overlayed in Image Studio software, so that both signals could be visualized simultaneously.

Multiplex Westerns

The two infrared fluorescent detection channels of the

Odyssey® Fc System enable simultaneous two-color analysis

– an advantage that is not available with chemiluminescent or

radioactive methods. Two primary antibodies are used, each

from a different host species (e.g., rabbit and mouse). IRDye®

secondary antibody conjugates (e.g., anti-rabbit and anti-mouse)

are then used to detect the primary antibodies and visualize the

targets.

Multiplex analysis makes normalization easy, and eliminates

error introduced by stripping and reprobing or by comparison of

separate blots. Superior image clarity and detail make it easier

to detect subtle mobility shifts caused by protein modifications

such as phosphorylation.

The Odyssey Fc System can detect Western blots in three ways:

n IR fluorescence at 700 nm

n IR fluorescence at 800 nm

n Chemiluminescence

Autofluorescent background can greatly influence detection

sensitivity. Low background increases the signal-to-noise

ratio, making faint signals detectable and extending the linear

dynamic range. At the IR wavelengths used by the Odyssey Fc

System, intrinsic membrane autofluorescence is extremely low.

At traditional visible wavelengths, membrane autofluorescence is

much higher and can mask weaker signals.

True multiplex Western blot detection requires excellent sensitivity

in both fluorescent channels – and only IR fluorescence can

achieve this sensitivity. Visible fluorescence typically provides

acceptable detection sensitivity in only one fluorescent channel.

Figure 9. Lysates of EGF-treated A431 cells were separated and trans-ferred to nitrocellulose. The blot was probed with anti-ERK and anti-phos-pho-ERK primary antibodies, and then detected with IRDye 680LT and IRDye 800CW secondary antibodies. Blot was imaged with Odyssey Fc System for 2 min. This phospho-ERK antibody crossreacts with phospho-EGFR (upper green band).

Membrane Autofluorescence and Multiplexing

Figure 10. Nitrocellulose and PVDF membranes were imaged at 700 and 800 nm wavelengths, then imaged at 532 and 635 nm (Cy3 and Cy5 chan-nels) with a GenePix® 4100A (Molecular Devices). Membrane background was dramatically reduced at IR wavelengths.

Mea

n M

emb

rane

Sig

nal

Wavelength

3000000

2500000

2000000

1500000

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500000

0800nm700nm532nm 635nm

PVDF

Nitrocellulose

Applications (cont.)

Gel Documentation

After electrophoretic separation of protein or nucleic acid samples, gels may be stained and imaged with the Odyssey Fc Imager.

Electrophoretic Mobility Shift Assays (EMSA)

EMSA is used to study protein-nucleic acid interactions. If the

protein binds to the nucleic acid sequence, this complex will

migrate more slowly on a gel – causing a mobility shift relative

to unbound nucleic acid. Radioactive labels have been typically

used for detection.

Figure 11. Purified fetuin protein was serially diluted (9.1 – 0.5 μg) and separated on a 10% gel. The gel was stained with IRDye Blue and imaged with Odyssey Fc in the 700 nm channel. Fetuin resolved as a doublet (2 min acquisition).

Figure 12. DNA samples (50 bp or 1 kb ladder) were pre-incubated with Syto 60 prior to gel loading. Samples were separated on a 1.2% agarose gel at 80V for 1 h. For each ladder, amounts loaded were 1, 0.5, 0.25, and 0.125 μg (left to right). Gel was imaged with Odyssey Fc in 700 nm channel (2 min acquisition).

Figure 13. EMSA gel with fluorescently labeled oligo. IRDye® 700 oligos with a p53 binding sequence were incubated for 30 min with nuclear extracts of serum-treated HeLa cells (0-10 μg). After electrophoresis on a 4-12% TBE gel, samples were imaged with the Odyssey Fc Imager for 2 min. As increas-ing amounts of nuclear extract were added, the change in amount of unbound oligo could be quantified.

Unbound IRDye OligoNuclear Extract (μg): 0 2.5 5 10

Oligo/Protein Complex

Unbound Oligo

IRDye end-labeled oligonucleotides and the Odyssey Fc System

are a safe, sensitive, and convenient alternative to radioactive

EMSA. Note: Gels must be < 10 cm x 12 cm.

IRDye® Blue (a colloidal Coomassie stain) is commonly used

to stain and view polyacrylamide protein gels. Coomassie is a

strong IR fluorophore, and can be imaged at 700 nm with the

Odyssey® Fc System to achieve superior sensitivity.

Syto® 60, a near-infrared fluorescent DNA stain, can be imaged at

700 nm – and is more sensitive than ethidium bromide. Protocols

for Syto 60 gel documentation and band excision can be found

at www.licor.com/syto60.

HeLa Nuclear Extract (μg)

Fluo

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ent

Sig

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40000

20000

0

R2 = 0.97144

2 4 6 8 10 12

Image Studio Software

With the Western and MPX™ Western (Multiplexer) Analysis Ribbons, you can draw your own shapes around the bands of interest,

automatically add shapes one at a time, or use the new automatic band finding feature. Data is presented in a table below the images,

for quick and easy export and printing. Custom reports give you as much or as little detail as you want. Build a customized lab book by

selecting the tables and image formats to be included, and edit the layout before printing.

Image Studio software is the new software platform for the

Odyssey Fc Imaging System. The intuitive ribbon format allows

for easy navigation. The user-friendly interface provides tabs

that move through the acquisition and analysis processes in

sequence. Hovering over an icon reveals detailed tool tips, to

help you learn the software quickly.

Image Acquisition is Easy1. Select the channel or channels to image (700 nm, 800 nm,

and/or chemiluminescence).

2. Select the acquisition time (with convenient time slider and custom options).

3. Click ‘Acquire’. A preview of the image will be shown during acquisition.

Figure 14. Western Blot Image Analysis with the Odyssey Fc System. A two-color Western blot was analyzed using the adjustable lane and automatic band finding features. Lanes and bands can be detected in both 700 and 800 nm channels simultaneously.

Before image analysis

Adjustable lane finding

Post band finding

MPX™ Blotting System

Western Reagents and Accessories

The MPX Blotting System is ideal for multiple-target Western

blot procedures that use PVDF or nitrocellulose membranes

(7 x 8.5 cm). It also allows you to quickly optimize experimental

conditions – blocking buffers, primary antibodies, and/or

secondary antibodies – in one easy experiment.

Low-volume channel ports (<160 μL) conserve antibodies

and reduce costs. Twenty-four channel ports are conveniently

spaced, staggered, and compatible with multi-channel pipettes.

Forty-eight targets can be visualized on a single membrane with

two-color detection. The MPX Blotting System is compatible with

many prep gels.

The LI-COR Biosciences IRDye infrared conjugates are optimized for a wide variety of applications. Odyssey Fc applications include

Western blots, MPX Westerns, In-Gel Westerns, EMSA, and gel documentation. For more information about LI-COR Biosciences

reagents and accessories designed to streamline your research, please view our online catalog or download our Products and Applica-

tions Guide at www.licor.com/bio.

FEATURES

n Screen a single sample and multiple targets on the same blot

n Screen up to 48 different targets with two-color detection

n Optimize blocking and antibody dilution parameters

n Conserve antibody and reagents

n Compatible with a variety of well configurations

n IRDye secondary antibody conjugates

n Blocking buffers

n Membranes

n Protein MW markers

Figure 15. MPX Blotting System and representative Western blot imaged with the Odyssey Fc shows evaluation of multiple primary and secondary antibody concentrations.

Available online at

www.licor.com/bio

n Gels

n Western blot detection kits

n Accessories

Related Products

Westerns on the Odyssey SystemsBetter Data, Cleaner Planet

Imaging with Odyssey Systems does not

require film, excessive water washes, or

hazardous chemicals associated with film

development.

For more details, visit:

www.licor.com/green

For more information on the Odyssey Fc Imaging System,

including applications and protocols, go to:

www.licor.com/odysseyfc.

� Odyssey® Fc Imaging Trays

� Odyssey Blocking Buffers

� IRDye® Infrared Dyes and Conjugates

� Blot Washer

� Western Incubation Boxes

� MPX Western Kit and Combos

� NucleoCounter® NC-100 for Automated Cell Counting

� Odyssey Pens

� Odyssey Soft Roller, 4 inches

Image Field Size:10 cm x 12 cm

Dynamic Range:22 bit (>6 logs)

Depth of Field for Best Sample Focus:6 mm

Patented FieldBrite™ XT Technology:CV <3%

Laser Lifetime: 20,000 hours

700 Channel Laser Source:Solid-state Laser Diode at 685 nm

800 Channel Laser Source:Solid-state Laser Diode at 785 nm

Detectors:Low-noise CCD. Thermoelectrically cooled.

Acquisition Times:• Fluorescence (700 and 800 nm) channels: 30 s,

2 min, 10 min plus variable time feature

• Chemiluminescence channel: 30 s, 2 min, 10 min, 60 min plus variable time adjustment feature

Focusing:None needed

Operating Conditions: For indoor use only; operating temperature 15-35°C and dewpoint < 22ºC, non-condensing; maximum operating temperature may be reduced at elevations above 2000 m.

Power Requirements:Universal input between 100-127 VAC (4 Amp) and 200-240 VAC (2 Amp); 50-60 Hz. Voltage fluctuations not to exceed 10% of the nominal voltage. Insulation Category II.

Sensitivity:Similar to industry-leading Odyssey® system.

Dimensions:41.4 cm W x 47 cm D x 67.3 cm H (16.3” W x 18.5” D x 26.5” H). Depth with imaging drawer open is 59.7 cm (23.5”).

Weight:27 kg (60 lb)

ETL/CETL/CE

System Specifications

References

1. Wang, Y.V., et al. Quantitative analyses reveal the importance of regulated Hdmx degradation for P53 activation, Proc Natl Acad Sci USA (2007) 104(30):12365-12370.

LI-COR is an ISO 9001 registered company. © 2010 LI-COR Inc. Specifications subject to change. LI-COR, Odyssey, MPX, FieldBrite, and IRDye are trademarks or registered trademarks of LI-COR, Inc in the United States and other countries. All other trademarks belong to their respective owners. The Odyssey Infra-red Imager, Odyssey Fc Imager, FieldBrite technology, and IRDye Infrared Dyes are covered by U.S. patents, foreign equivalents, and other patents pending.

The LI-COR board of directors would like to take this opportunity to return thanks to God for His merciful providence in allowing LI-COR to develop and commercialize products, through the col-lective effort of dedicated employees, that enable the examination of the wonders of His works.

“Trust in the LORD with all your heart and do not lean on your own understanding. In all your ways acknowledge Him, and He will make your paths straight.”—Proverbs 3:5,6

980-11302 06/10

U.S.

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LI-COR Biosciences GmbH Siemensstraße 25A D-61352 Bad Homburg Germany Phone: +49 (0) 6172 17 17 771 Fax: +49 (0) 6172 17 17 799 Email: gmbh@licor.com

LI-COR Ltd., UKServing UK, Ireland, and Scandinavia

LI-COR Biosciences UK LtdSt. John’s Innovation Centre Cowley Road Cambridge CB4 0WS United Kingdom Phone: +44 (0) 1223 422104 Fax: +44 (0) 1223 422105 Email: UK@licor.com

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View a complete list of our international distributors at:

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