5X RIPA Buffer Reagent

Store RIPA Buffer concentrate at room temperature.. Shelf Life: 1 year from date of receipt under proper storage conditions; aliquot to avoid multiple freeze thaw cycles

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 degree C for up to 6 months.

20 mL

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 (56.7 cm2), 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 degree C. Samples may be frozen at -80 degree 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 degree C. 7. Centrifuge for 10 minutes (14000 x g).
8. Transfer the supernatant to a clean tube and maintain at 4 degree C. Samples may be frozen at -80 degree C.

Cell Signaling and Protein Biology; Protein Analysis Tools; Protein Isolation and Extraction; RIPA Buffer