5X RIPA Buffer with Protease Inhibitor Cocktail

• Buffer is able to extract cytoplasmic, membrane, and nuclear proteins
• Suitable for downstream assays such as reporter assays, protein assays, immunoassays and protein purification
• Protease Inhibitor Cocktail included

1X RIPA Buffer: Mix the 5X RIPA Buffer by inverting/vortexing the bottle a few minutes. Dilute the 5X RIPA Buffer to 1X with deionized water. Stir to homogeneity. Store at 4 °C for up to 6 months. Protease Inhibitor Cocktail: Immediately before use dilute the Protease Inhibitor Cocktail 1:100 with 1X RIPA Buffer. Stir to homogeneity. Do not store diluted solutions containing protease inhibitors.

I. Adherent Cells

1. Culture cells to approximately 80-90 % confluence.
2. Aspirate the culture media and wash twice with cold PBS.
3. Add cold 1X RIPA Buffer to the cells, spreading evenly. Use 1 mL of buffer per 10-cm dish 2 (56.7 cm ), maintaining on ice for 5 minutes. Cells may be scraped with a cell scraper.
4. Collect the lysate into a microcentrifuge tube.
5. Centrifuge for 10 minutes (14000 x g).
6. Transfer the supernatant to a clean tube and maintain at 4 °C. Samples may be frozen at -80 °C.

II. Suspension Cells

1. Collect the cells into an appropriate conical centrifuge tube.
2. Centrifuge for 5 minutes (600 x g).
3. Remove and discard the supernatant.
4. Wash the cells twice with cold PBS, centrifuging for 5 minutes between (600 x g).
5. Add cold 1X RIPA Buffer to the cell pellet. Use 1 mL of buffer per 40 mg of wet cell pellet. Gently resuspend the pellet by pipetting up and down.
6. Shake the suspension for 15 minutes at 4 °C.
7. Centrifuge for 10 minutes (14000 x g).
8. Transfer the supernatant to a clean tube and maintain at 4 °C. Samples may be frozen at -80 °C.