RIPA Buffer (RIPA-50)

Strong detergent lysis buffer
pH 7.4, 10X solution
Use to solubilize menbrane proteins and release cytosolic components
Convenient pre-formulated solution.

Cell Lysis Protocol:

Note: RIPA buffer (Part No. RIPA-50) is provided as a 10X solution. Dilute to 1X with dH20 prior to your experiment.

A. Cell Culture:

It is recommended that cells are cultured to 80-90% confluency prior to performing cell lysis. Cells should be washed free of serum proteins using PBS to prevent appearance of non-specific serum protein bands in downstream Western blots. Remove PBS prior to the addition of RIPA buffer.

B. Cell Lysis:

Dilute sufficient 10X RIPA buffer in dH20 to make a 1X solution. Cool RIPA buffer in ice and add protease inhibitors and phosphatase inhibitors (if required) immediately prior to cell lysis. We recommend using 300 ul of 1X RIPA Buffer solution for one to three 10 cm cell culture dishes of cells. Scale accordingly for other numbers or sizes of cell culture dishes according to the surface area of the dish.

1. Lyze cells and generate a supernatant fraction as follows: Apply the ice cold RIPA Buffer solution to cells for 15 minutes: Use 300 ul 1X RIPA Buffer for one to three 10 cm plates of cells. Scale accordingly for other numbers or sizes of cell culture dishes according to the surface area of the cell culture dish.

A simple method to dislodge and lyze adherent cells is to place the washed (and PBS removed) cell culture plates on a bed of ice. Dispense ice cold 1X RIPA Buffer with protease/phosphatase inhibitors over the cell layer, rotate the plate by hand to cover cells with a film of RIPA Buffer, then immediately dislodge the cells with a cell scraper. This cell suspension can also be transferred sequentially over multiple cell culture plates to collect a concentrated cell suspension. Next, use a transfer pipette to siphon the cells into a 1.5ml microcentrifuge tube.

Tap the tube with the cell suspension vigorously several times to lyze membranes. You can also vortex the cell suspension to lyze membranes if the immediately downstream application is Western Blotting (do not vortex if the downstream application is immunoprecipitation). Leave the cell suspension on ice for 15 min.

2. Centrifuge the cell lysate in a cooled microcentrifuge at full speed for 15 min to partition supernatant and pellet. Collect the supernatant fraction, which contains extracted membrane and cytosolic proteins. Dispense this supernatant into another 1.5 ml microcentrifuge tube that is placed in ice. The supernatant can be aliquoted and stored at -20C, or -80C for longer term storage.