Dear fellow biochemists,

I need some advice on Western Blotting, more specifically the use of certain protease inhibitors with the RIPA cell lysis buffer and protease inhibitor cocktail. A Millipore technical representative recommended my using "539134 | Protease Inhibitor Cocktail Set III, EDTA-Free - Calbiochem" as a good general protease inhibitor cocktail.

I have another protease inhibitor cocktail which includes: AEBSF, bestatin, E-64, and pepstatin A (E.coli protease inhibitor cocktail) but it does not include the aprotinin or the leupeptin inhibitors. Is there a risk of proteolysis in a mammalian cell lysate without these latter 2 inhibitors? The specific cell types which we are using are: HUVEC (human umbilical vein endothelial cells) and HT-29 adenocarcinoma cell line.

Dr Robert Weinberg
Dept of Biology, MIT
email: rweinber@mit.edu
fax: 617-977-0902

These cocktails are not very specific nor is there a perfect combo that completely reduces protease activity (and does not cause downstream issues).

For proteomic applications we found that working quickly on ice results in little or no detectable degradation. That being said, either of the mixes would cover the range of major protease classes.