rpwdoc

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