When to use PMSF in a cell lysis buffer? - (Oct/14/2008 )

I usually use Sigma P2714, a protease inhibitor mixture, for general use. I noticed that many times, people included PMSF in lysis buffer. Could anybody explain to me when to use PMSF (depending on cell type, tissue type, bio-endpoint you are going to focus on, cellular component you want to get, or what? )? It seems that Sigma suggests a different product for getting membrane proteins, but the difference is not regarding PMSF.
Thanks in advance!
Hanhan

I noticed the summary post already included PMSF, but still hope my specific question could attract interest . Simply put: could other serine protease inhibitors (like those in a Sigma product) replace PMSF?

PMSF is protease inhibitor. So it should be used as soon as possibly to avoid protease action. Basically protease are blocked in the cell (endosomes of i'm not wrong). So when you lyse cell and membranes, protease are relased. So you shold inhibit them.

For membrane protein, maybe the solubility of the solutions used are uncompatible with PMSF and/or some other products are better than this general one for this kind of protein extraction.

Thanks a lot! So PMSF is good for protecting soluble proteins like those in cytosol.

I use PMSF in combination with the sigma protease inhibitor cocktail mix for every cell lysate I prepare. I also add sodium orthovanadate and sodium fluoride to protect the phospho-status of my proteins.

Just wondering if you know you have to activate your sodium orthovanadate with a series of boiling and pHing with HCl? I've been using it for years and didn't realize this. Now I just buy a commercial cocktail, much like the Roche complete protease inhibitor cocktail. I also add AEBSF from sigma. It's the same as PMSF but much more stable.

yeah - the activation is damn annoying... my series is somewhat more like a few times, but you see it turn clear.

Regarding the use of PMSF during cell lysis, which is the best solvent to have the PMSF in? Previously we used to have PMSF in ispropanol. But the lab where I am right now in, they use PMSF in ethanol. So does it make any difference? Just wondering.

rigzin

DMSO is also a good solvent to stabilize PMSF