RIPA Buffer (10X) #9806
RIPA buffer is used to lyse cells and tissues.
Directions for Use
- If buffer will be continually used, it is recommended that the 10x buffer be kept at 4°C for 1-2 weeks. For longer periods of time, buffer should be stored at -20°C. Aliquotting of 10x buffer is recommended if many small experiments are to be performed.
- Thaw 10x buffer at 24-30°C, mixing end-over-end.
- Dilute 10x buffer in Milli-Q water or water of equivalent quality.
- Chill 1x buffer on ice and add PMSF just prior to use.
For lysis of adherent cells, we recommend the following: (all reagents and lysates must be kept cold).
- Treat cells as desired.
- Wash plate with PBS to remove residual media.
- Add 400 ul of 1x RIPA buffer/10 cm dish.
- Incubate plate on ice for 5 minutes.
- Scrape cells.
- Sonicate briefly.
- Centrifuge extract for 10 minutes at 14,000 x g in a cold microfuge.
- Remove supernatant for use.
- For non-adherent cells, add 400 ul of buffer per 107 cells once they have been washed in 1X PBS and pelleted.
- The CST 1X RIPA Buffer can be used for lysis of tissue samples, although a homogenization step is recommended after adding lysis buffer. Extract the tissue at a ratio of 100 mg of tissue to 1 ml of buffer. Sonication of the tissue lysate is also required.
- Additional protease inhibitors can be added to the lysis buffer without any difficulties.
Solutions and Reagents
- 1X RIPA Buffer:
- 20 mM Tris-HCl (pH 7.5)
- 150 mM NaCl
- 1 mM Na2EDTA
- 1 mM EGTA
- 1% NP-40
- 1% sodium deoxycholate
- 2.5 mM sodium pyrophosphate
- 1 mM beta-glycerophosphate
- 1 mM Na3VO4
- 1 µg/ml leupeptin
Note: CST recommends adding 1 mM PMSF immediately before use.
- Kong, B. et al. (2010) Oncogene 29, 5146-58. Applications: Western Blotting
- Wang, Z. et al. (2011) Autophagy 7, 401-11. Applications: IP
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
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- 5871 Protease Inhibitor Cocktail (100X)
- 5872 Protease/Phosphatase Inhibitor Cocktail (100X)
- 7780 BCA Protein Assay Kit
For Research Use Only. Not For Use In Diagnostic Procedures.