PRODUCT INFORMATION SHEET

RIPA Lysis and Extraction Buffer

Catalog Numbers 89900 and 89901
Doc. Part No. 2161782 Pub. No. MAN0011565 Rev. B.0
WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.

Contents and storage

Cat. No. Description Storage

89900 RIPA Buffer, 100 mL Upon receipt, store at 4°C. Product shipped at ambient
temperature.
89901 RIPA Buffer, 250 mL
Note: Product shipped at ambient temperature.
Contents: 25 mM Tris•HCl pH 7.6, 150 mM NaCl, 1%
NP-40, 1% sodium deoxycholate, 0.1% SDS

Product description
™
The Thermo Scientific RIPA buffer is one of the most reliable buffers used to lyse cultured mammalian cells from both plated cells and
cells pelleted from suspension cultures. This buffer enables protein extraction from cytoplasmic, membrane and nuclear proteins and is
compatible with many applications, including reporter assays, protein assays, immunoassays and protein purification.

Guidelines
™ ™
• RIPA Buffer does not contain protease or phosphatase inhibitors. If desired, add protease inhibitors, such as Thermo Scientific Halt
™
Protease Inhibitor Cocktail (Cat. No. 78410) and Halt Phosphatase Inhibitor Cocktail (Cat. No. 78420) to the reagent to prevent
proteolysis and maintain phosphorylation status of proteins. Add protease and phosphatase inhibitors immediately before use.
• Use 1mL of cold RIPA Buffer for every 5 × 106 of HeLa or A431 cells (∼20 µL of packed cells, which is equivalent to ∼40 mg of cells).
To obtain concentrated protein extracts, directly lyse cells on plate and use less buffer.
• Some protein kinases and other enzymes may be sensitive to the components of the RIPA Buffer, resulting in their decreased activity.
In such cases, prepare a RIPA buffer that does not contain sodium deoxycholate and SDS.
™ ™
• RIPA Buffer is compatible with the Thermo Scientific Pierce BCA Protein Assay Kit (Cat. No. 23225).

Lyse Monolayer-cultured mammalian cells

Note: If desired, add protease and phosphatase inhibitors to the RIPA Buffer immediately before use.

1. Carefully remove (decant) culture medium from adherent cells.

2. Wash cells twice with cold PBS.

3. Add cold RIPA Buffer to the cells. Use 1 mL of buffer per 75 cm2 flask containing 5 × 106 HeLa or A431 cells. Keep on ice for
5 minutes, swirling the plate occasionally for uniform spreading.

4. Gather the lysate to one side using a cell scraper, collect the lysate and transfer to a microcentrifuge tube. Centrifuge samples at
∼14,000 × g for 15 minutes to collect the cell debris.
Note: To increase yields, sonicate the pellet for 30 seconds with 50% pulse.

5. Transfer supernatant to a new tube for further analysis.

For Research Use Only. Not for use in diagnostic procedures.

Lyse Suspension-cultured mammalian cells
Note: If desired, add protease and phosphatase inhibitors to the RIPA Buffer immediately before use.

1. Collect cells by centrifugation at 2500 × g for 5 minutes. Discard the supernatant.

2. Wash cells twice in cold PBS. Collect cells by centrifugation at 2500 × g for 5 minutes.

3. Add RIPA Buffer to the cell pellet. Use 1 mL of RIPA buffer for 40 mg (∼5 × 106 of HeLa cells) of wet cell pellet. Pipette the mixture up
and down to suspend the pellet.
Note: To increase yields, sonicate the pellet for 30 seconds with 50% pulse.

4. Shake mixture gently for 15 minutes on ice. Centrifuge mixture at ∼14,000 × g for 15 minutes to pellet the cell debris.

5. Transfer supernatant to a new tube for further analysis.

Troubleshooting and FAQs

Visit our online FAQ database for tips and tricks for conducting your experiment, troubleshooting information, and FAQs. The online FAQ
database is frequently updated to ensure accurate and thorough content.
• For troubleshooting information and FAQs for this product: https://www.thermofisher.com/ripabufferfaqs
• To browse the database and search using keywords: thermofisher.com/faqs

Related Thermo Scientific™ products

Cat. No. Product

78410 Halt™ Protease Inhibitor Cocktail Kit

78420 Halt™ Phosphatase Inhibitor Cocktail, 1mL

78248 B-PER™ Bacterial Protein Extraction Reagent, 500mL

78990 Y-PER™ Yeast Protein Extraction Reagent, 500mL

89826 Mem-PER™ Membrane Protein Extraction Reagent Kit

78833 NE-PER™ Nuclear and Cytoplasmic Extraction Kit

23227 Pierce™ BCA Protein Assay Kit

26148 Pierce™ Direct IP Kit

34080 SuperSignal™ West Pico Chemiluminescent Substrate, 500mL

34076 SuperSignal™ West Dura Extended Duration Substrate, 200mL

General references
Cao, F., et al. (2005). Identification of an essential molecular contact point on the duck hepatitis B virus reverse transcriptase. J Virol 79(16):
10164-70.
Pfrepper, K.I. and Flugel R.M. (2005). Molecular characterization of proteolytic processing of the gap proteins of human spumaretrovirus.
Methods in Mol Biol 304:435-44.
Sefton, B.M. (2005). Labeling cultured cells with 32Pi and preparing cell lysates for immunoprecipitation. Unit 18.2. F. M. Ausubel, R. Brent,
R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl (eds.) Current Protocols in Molecular Biology. John Wiley & Sons, Inc.

Documentation and support

Customer and technical support
Visit thermofisher.com/support for the latest service and support information.
• Worldwide contact telephone numbers
• Product support information
– Product FAQs
– Software, patches, and updates

2 RIPA Lysis and Extraction Buffer Product Information Sheet

 – Training for many applications and instruments
• Order and web support
• Product documentation
– User guides, manuals, and protocols
– Certificates of Analysis
– Safety Data Sheets (SDSs; also known as MSDSs)
Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer.

Limited product warranty

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Conditions of Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact
Life Technologies at www.thermofisher.com/support.

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The information in this guide is subject to change without notice.

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Revision history: Pub. No. MAN0011565
Revision Date Description
B.0 28 January 2020 Moved troubleshooting content to thermofisher.com.

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