My protein sample is extracted with RIPA + PMSF ( Phenylmethylsulfonyl fluoride). After protein assay, the concentration is too high (>900ug/mL) for direct application for gelatin zymography (non-reducing protein). I want to prepare a diluted stock (100uL) that can be used for zymography, and can be safely stored in -80 for later usage. What will be a good diluent?
Thank you.
First, PMSF is not an inhibitor, but an inactivator of Serineproteases, that is, it permanently modifies the catalytic serine and thereby sends the enzyme to kingdom come. I would not use it for isolation of proteases (unless I am only after enzymes of different classes).
To prevent unspecific adsorption of dilute proteins on vessel walls you could try polyvinylpyrolidone (PVP). 1% is used for blocking membranes in westerns, for your purpose 0.1% should do.
If you are looking at matrix metalloproteinases via gelatin zymography, keep the non-MMP protease inhibitors. Since you have so much ptotein, why not try a comparison of storage in your lysis buffer and gel sample buffer. The problem is that the inhibitors precipitate and the sample must be gently warmed to redissolve PMSF. there are big stability differences for different tissues/cell lines.
Ellen A G Chernoff : PMSF is unstable in aqueous solutions, lasts about 20 min. Since it is an inactivator, it should have done its job by then. Stock solutions of PMSF should be prepared in aprotic solvents like isopropanol (but not in DMSO, which can take dissolved substances through intact skin).
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