RIPA Buffer (RIPA-50)

Background:  RIPA buffer is commonly used to extract proteins for immunoprecipitation and/or Western blotting.  RIPA has strong extraction properties that solubilizes and extracts proteins from membranes and releases cytoplasmic cellular components.  RIPA buffer is mildly denaturing since it contains ionic detergent.  FIVEphoton Biochemicals offers pre-made 5X RIPA buffer in 30 ml aliquots. 

Contents:  5X RIPA

250 mM Tris pH 7.4

0.75 M NaCl

5 mM EDTA

0.5% SDS

5% NP-40

2.5% sodium deoxycholate

Protocol

 

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 5X 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 µl 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 µl 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 -20^oC, or -80^oC for longer term storage.

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Product Citation

       Tsai, M. H., Lin, Z. C., Liang, C. J., Yen, F. L., Chiang, Y. C., & Lee, C. W. (2014). Eupafolin inhibits PGE2 production and COX2 expression in LPS-stimulated human dermal fibroblasts by blocking JNK/AP-1 and Nox2/p47phox pathway. Toxicology and applied pharmacology, 279(2), 240-251.

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Safety:  Irritant.  Wear eye protection and gloves.  Avoid skin and eye contact and ingestion.

Storage:  RT.