Hi there,

I think it is actually much better to supplement the lysis buffer with protease inhibitors right before use. These are not meant to be  stable for a long time at their working concentrations and freeze/thaw cycle might also affect their stability too.

I totally agree with Dominique. Aliquot 100x inhibitor mixes instead.

We use PMSF as protease inhibitor. The PMSF stock is in ethanol and stored in -20 freezer. PMSF will degrade very fast at room temperature in lysis buffer, probably half an hour. So we always add inhibitors right before use.

some inhibitors are ok, like EDTA, phosphoramidon, soybean trypsin inhibitor, etc, whereas PMSF and other electrophilic inhibitors are not. We kept inhibitors in DMSO stocks and added right before use. I don't think E64 is stable either in aqueous solution. So your answer is that it depends on what type of inhibitors you need to use. If you don't know, and want to use commercially available cocktails, then no you can't store in lysate buffer.

I also agree about keeping aliquots of 100x inhibitor and adding to lysis buffer fresh before your experiment.