Minute™ Total Protein Extraction Kit for Animal Cultured ......Minute™ Total Protein Extraction Kit for Animal Cultured Cells and Tissues Catalog #: CLT-SD-001/SN-002, Shipping

Protein Extraction Kit Cell Lab Tech, Inc.

Minute™ Total Protein Extraction Kit for Animal

Cultured Cells and Tissues Catalog #: CLT-SD-001/SN-002, Shipping Weight: 0.5lbs

Minute™ total protein extraction kit for

animal cultured cells and tissues is

composed with optimized cell lysis

buffer and protein extraction filter

cartridges with 2.0 ml collection tubes.

The kit is designed to rapidly extract

total proteins from animal cells and

tissues (invertebrate and vertebrate) for

protein analysis and further purification.

Since protein profiles extracted by

denaturing and native cell lysis buffer are not identical, for a given

application one might be superior to the other. This kit provides both

denaturing and native cell lysis buffers for users to test and select the best

one for a specific application. Due to the use of the protein extraction filter

cartridges, the extraction volume can be as small as 20 µl and as large as

500 ul. This unique feature is very useful in situations where available

starting material is a limiting factor. Total proteins can be extracted from

cultured cells/tissues in 1-8 min with high yield (2-8 mg/ml). Extracted

proteins can be use as a good starting material for protein analysis and

small scale protein purification in column chromatography.

4062 Fabian Way, STE 2B, Palo Alto, CA 94303 Tel: 408-886-8592 Fax: 408-465-5299 email: [email protected] www.cell-labtechcom

MinuteTM Total Protein Extraction Kit for Animal Cultured Cells and Tissues User Manual Cell Lab Tech Inc.

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MinuteTM Total Protein Extraction Kit for Animal Tissues or Cultured Cells

Catalog Number: CLT-SD-001/CLT-SN-002

_______________________________________________________________________ Description Invent Biotechnologies MinuteTM total protein extraction kit for animal tissues or cultured cells is composed of optimized cell lysis buffer and protein extraction filter cartridges with 2.0 ml collection tubes. The kit is designed to rapidly extract total proteins from animal tissues or cultured cells (invertebrate and vertebrate) for protein analysis and further purification. Since protein profiles extracted by denaturing and native cell lysis buffer are not identical, for a given application one lysis buffer might be superior to the other. This kit provides both denaturing and native cell lysis buffers for users to test and select the best one for a specific application. Due to the use of the protein extraction filter cartridges, the extraction volume can be as small as 20 µl and as large as 500 µl. This unique feature is very useful in situations where available starting material is a limiting factor. Total proteins can be extracted from tissues/cultured cells in 1-8 min with high yield (2-8 mg/ml). Application MinuteTM total protein extraction kit is designed to rapidly extract total proteins from invertebrate and vertebrate tissues or cultured cells for applications such as SDS-PAGE, immunoblottings, IP, ELISA, enzyme assays and other applications. This kit provides the most rapid method currently available for preparation of whole cell protein extract. Extracted proteins can also be use as a good starting material for small scale protein purification in column chromatography. Buffer Formulation: Proprietary Kit components

1. 25 ml denaturing cell lysis buffer (SD-001) 2. 25 ml Native cell lysis buffer (SN-002) 3. 50 protein extraction filter cartridges 4. 50 collection tubes with cap 5. Plastic rods (4)

Storage Store native cell lysis buffer (SN-002) at 4oC and rest of the kit at room temperature


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Important Product Information The MinuteTM total protein extraction kits are designed to extract total protein rapidly. The use of protease inhibitors is not necessary prior to extraction. However if downstream application takes significant amounts of time or the protein extract will be stored for longer period of time, addition of protease inhibitors to cell lysis buffer is recommended. For determination of protein concentration, BCA kit (Pierce) is recommended. To study protein phosphorylation, phosphatase inhibitors (such as PhosStop from Roche) should be added to lysis buffer prior to use. Additional Materials Required 1 X PBS Vortexer Table-Top Microcentrifuge BCA Protein Assay Kit (Pierce, Cat #. 23227)


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Protocols: Total Protein Extraction for Animal Tissues (Part I) Denaturing Total Protein Extraction (SD-001)

Following procedures are for 15-20 mg starting animal tissues. For insect such as Drosophila use 15- 20 larva, pupa or adult flies/sample. If smaller or larger amounts of starting materials are used adjust the amount of cell lysis buffer proportionately.

1. Prior to protein extraction, pre-chill the protein extraction filter cartridge in

collection tube on ice. 2. Place 15-20 mg fresh/frozen tissue in the filter. Grind the tissue with a plastic rod

for 50-60 time with twisting force, add 200 µl denaturing cell lysis buffer to the filter and continue to grind for 30-60 times. Note: The plastic rod is reusable. For cleaning, rinse it thoroughly with distilled water and dry it with paper towel.

3. Cap the filter and incubate at room temperature for 1-2 min. Centrifuge at a microcentrifuge at top speed for 1-2 min. The supernatant of flow through contains denatured total protein extract.

Important Note: the presence of small amount of un-lysed tissue will not affect the quality of the samples

Native Total Protein Extraction (SN-002)

1. Prior to protein extraction, pre-chill the protein extraction filter cartridge in collection tube on ice.

2. Place 15-20 mg fresh/frozen tissue in the filter. Grind the tissue with a plastic rod for 50-60 time with twisting force, add 200 µl native lysis buffer (SN-002) to the filter and continue to grind for 30-60 times.

Note: The plastic rod is reusable. For cleaning, rinse it thoroughly with distilled water and dry it with paper towel.

3. After tissue grinding, incubate the tube on ice with cap opening for 5 min. Cap the filter and centrifuge in a microcentrifuge at top speed for 1-2 min at 4oC. The supernatant of flow through contains native total protein extract.


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Total Protein Extraction for Cultured Cells (Part II) Denaturing Total Protein Extraction (SD-001)

A. Non-Adherent Cells 1. Prior to protein extraction, pre-chill the protein extraction filter cartridge with

collection tube on ice. 2. Harvest cells by low speed centrifugation. Wash the cells in cold PBS once in a

1.5 ml microcentrifuge tube and pellet the cells by centrifugation at 3000 rpm for 2-3 min. Aspirate the supernatant and leave small amount of PBS (about the volume of packed cells) in the tube. Vortex the tube briefly to resuspend the cells.

3. Add appropriate amounts of cell lysis buffer to the cell suspension (Table 1), vortex briefly to lyse the cells.

Important Note: the presence of small amount of un-lysed cells woµld not affect the quality of the samples.

4. Transfer/pour the cell lysate to pre-chilled filter cartridge(s) in collection tube(s)

and centrifuge in a microcentrifuge for 30 seconds at top speed (14,000-16,000 rpm).

5. Immediately place the collection tube on ice. Discard the filter cartridge according to your institution’s waste disposal protocol. The cell lysate is now ready for downstream applications.

Table 1 Lysis Buffer Volume for Different Packed Cell Volumes*

Packed cell volume (µl) lysis buffer (µl) Equivalent cell # X 106

3 20 0.3 5 50 0.5 10 100 1 20 200 2 40 500 3

*For NIH3T3 and 293T cells 10 µl packed cell volume is equivalent to about106 cells

B. Adherent cells

1. Prior to protein extraction pre-chill the protein extraction filter cartridge (placed in collection tube) on ice.

2. Grow adherent cells to 90-100% confluence and wash the cells once in the tissue culture plates, dishes or flasks with cold PBS, aspirate the buffer completely.


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3. Add appropriate amounts of cell lysis buffer (Table 2), swirl to distribute the lysis buffer over the entire surface of tissue cultures. Scrape the lysed cells with a pipette tip or a transfer pipette and transfer the cell lysate to pre-chilled protein extraction filter cartridge(s) in collection tub(s). Centrifuge at top speed (14,000-16.000 rpm) in a microcentrifuge for 30 seconds.

4. Immediately place the collection tube on ice. Discard the filter cartridge according to your institution’s waste disposal protocol. The cell lysate is now ready for downstream applications.

Table 2 Amounts of lysis buffer required for different amount of adherent cells

Containers Approximate Cell# Lysis buffer(µl)

24-well plate 0.1-0.2 Million 50 6-well plate 0.6-0.8 Million 200 25 cm2 flask 1.5-2 Million 500

Native Total Protein Extraction (SN-002)

A. Non-Adherent Cells 1. Prior to protein extraction pre-chill native cell lysis buffer (SN-002) and protein

extraction filter cartridge with collection tube on ice. 2. Harvest the cell by low speed centrifugation. Wash the cell in cold PBS once and

pellet the cells by centrifugation at 3000 rpm for 2-3 min. Aspirate the supernatant and leave small amount of PBS (about the volume of packed cells) in the tube. Vortex briefly to resuspend the cells.

3. Add appropriate amounts of lysis buffer to the cell suspension (Table 3) and vortex the tube vigorously for 15 seconds. Place the tube on ice for 3-5 min and vortex vigorously for 10 seconds.

4. Transfer/pour the cell lysate to pre-chilled filter cartridge, cap the tube and centrifuge in a microcentrifuge for 30 seconds at 14,000-16,000 rpm.

5. Immediately place the collection tube on ice and discard the filter cartridge according to your institution’s waste disposal protocol. The cell lysate is now ready for downstream applications.

Table 3 Lysis buffer volume for different packed cell volumes*

Packed cell volume (µl) lysis buffer (µl) Equivalent cell# X 106

3 25 0.3 5 50 0.5 10 100 1 20 200 2 40 500 3

*For NIH3T3 and 293T cells 10 µl packed cell volume is equivalent to about106 cells


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B. Adherent cells 1. Prior to protein extraction pre-chill native cell lysis buffer (SN-002) and the

protein extraction filter cartridge with collection tube on ice. 2. Grow adherent cells to 90-100% confluence and wash the cells twice in the tissue

culture plates, dishes or flasks with PBS, aspirate the buffer completely. 3. Add appropriate amounts of lysis buffer (Table 4), swirl to distribute the lysis

buffer over the entire surface of tissue cultures. Place the tissue culture on ice for 5 min. Scrape the lysed cells with a pipette tip or with a transfer pipette and transfer cell lysates to pre-chilled protein extraction filter cartridge(s), centrifuge at 14,000 to 16,000 rpm in a microcentrifuge for 30 seconds..

4. Immediately place the collection tube on ice and discard the filter cartridge according to your institution’s waste disposal protocol. The cell lysate is now ready for downstream applications.

Table 4 Amounts of Lysis Buffer Required for Different Amount of Adherent Cells

Containers Approximate Cell# Lysis buffer((µl)

24-well plate 0.1-0.2 Million 50 6-well plate 0.6-0.8 Million 250 25 cm2 flask 1.5-2 Million 500


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Troubleshooting Problem Solution The lysate is too viscous to pipette with a 200-1000 µl pipette tip

Pour the lysate into protein extraction filter cartridge

Retention of cell lysate in protein extraction filter cartridge after 30 seconds of centrifugation

Decrease amounts of starting cells/tissues or increase amount of lysis buffer

Low protein concentration Low protein band intensity at high molecular weight range (100-300 KDa)

Increase amounts of cells/tissues or decrease amount of cell lysis buffer Increase amount of lysis buffer and make sure cells/tissues are completely lysed.

Invent Biotechnologies, Inc. Material Safety Data Sheet 8961Aztec Drive Emergency Phone 952-400-6898 Eden Prairie, MN 55347, USA Fax 952-400-6889 E-Mail [email protected]

SECTION 1 — CHEMICAL PRODUCT AND COMPANY IDENTIFICATION Product Name Catalog # MinuteTM Bacterial Total Protein Extraction Kit SB-004 MinuteTM Detergent-Free Protein Extraction Kit (For Animal Cultured Cells and Tissues) SN-006 SECTION 2 — COMPOSITION/INFORMATION ON INGREDIENTS Hazardous/ Non-hazardous Components Phosphate-buffered saline (10%) SECTION 3 — HAZARDS IDENTIFICATION Physical/chemical Hazards Harmful Human health hazards Harmful if swallowed, irritating to eye and skin NFPA rating (1-4) Health rating: 2, Reactivity rating: 1, contact rating: 3 SECTION 4 — FIRST AID MEASURE Inhalation If inhaled, remove to fresh air. If breathing is difficult, give oxygen and seek medical attention. Ingestion Do not induce vomiting unless directed to do so by medical personal. If large quantities of this material are swallowed, call a physician immediately. Skin contact In case of contact, immediately flush the skin with plenty of water Remove contaminated clothes and shoes. Eye contact In case of contact, immediately flush eyes with plenty of water for at least 15 min. get medical attention. Aggravating condition Prolonged exposure is not known for aggravate medical condition. SECTION 5 — FIRE FIGHTING MEASURES Flammability May be combustible at high temperature. Extinguishing media Suitable Use dry chemical for small fire, for large fire, use water spray, fog

or foam. Do not use water jet Hazardous Decomposition These products are carbon oxides (CO, CO2), nitrogen oxides etc. Fire-fighting procedure Fire fighter should ware positive pressure self-contained breathing Apparatus and full turnout gear. Protecting of fire-fighters Be sure to use approved/certified respirator or equipment.

SECTION 6 — ACCIDENTAL RELEASE MEASURE Personal Precautions Splash goggles, full suit, boots and gloves. Consult a specialist. Spill and leak Use appropriate tools to put the spilled solid in a convenient waste disposal container. SECTION 7 — HANDLING AND STORAGE Handling Keep away from heat and source of ignition. Empty containers

pose a fire risk. Storage Keep container tightly closed and store at room temperature. SECTION 8 — EXPOSURE CONTROL / PERSONAL PROTECTION Engineering measures Use process enclosure, local exhaust ventilation, or other Engineering controls to keep airborne level recommended limits. Hygiene measures Wash hands after handling the materials. Personal protective equipment Skin and body Lab coat Eyes Safety glasses SECTION 9 — PHYSICAL AND CHEMICAL PROPERTIES Physical state liquid Color Not available Molecular Weight Not available Solubility Not available Flash point Not available Explosive properties Not available SECTION 10 — STABILITY AND REACTIVITY Stability The product is stable Conditions to avoid Not available Products of hazardous Decomposition These products are carbon oxides (CO, CO2), nitrogen oxides etc. SECTION 11 — TOXICOLOGICAL INFORMATION RTECS # N/A Local effects Skin irritation Not available Acute toxicity LD50 and LC50: Not available Chronic toxicity Repeated or prolonged exposure is not known for aggravate

medical condition. Other Toxic Effects on humans Not available Carcinogenic effects Not available Mutagenic effects Not available Reproduction toxicity Not available Teratogenic effects Not available

SECTION 12 — ECOLOGICAL INFORMATION Ecotoxicity Not available Toxicity of product of biodegradation The product itself and its degradation products are not toxic. SECTION 13 — DISPOSAL CONSIDERATIONS Methods of disposal Waste must be disposed of in accordance with federal, state, and local control regulations. SECTION 14 — TRANSPORT INFORMATION IATA Proper shipping name Not classified as dangerous in the meaning of transport regulations. Hazard class Not available Subsidiary class Not available Packing group Not available Un-no Not available SECTION 15 — REGULATORY INFORMATION EU regulation

Risk phrases This product is not classified according to the EU regulation. U.S. Federal Regulations This product is not regulated by SARA 313 This product does not contain HAPs. HCS Classification Not-controlled under the HCS (United States) State regulations California Proposition 65 This product does not contain chemicals listed under proposition 65 WHMIS (Canada) Non-controlled SECTION 16 — OTHER INFORMATION These products are sold for research use only. They are not intended for human or animal therapeutic or clinical diagnostic use. They are not intended for food, drug, household or cosmetic use. A technically competent individual must supervise the use of these products. The above product information was acquired by diligent search and/or investigation and the recommendations are based on prudent application of professional judgment. The information is to be used as a guide and shall not be taken as being all inclusive. All materials and mixtures may be present unknown hazards and should be used with caution. Since Invent Biotechnologies Inc. cannot control the actual methods or conditions of use, the company shall not held responsible for any damages or losses resulting from contact with the product described herein. The information in this msds does not constitute a warranty, express or implied, including any implied warranty of merchantability or fitness for any particular purpose. End of Safety Data Sheet

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Minute™ Total Protein Extraction Kit for Animal Cultured ......Minute™ Total Protein Extraction Kit for Animal Cultured Cells and Tissues Catalog #: CLT-SD-001/SN-002, Shipping