Use of protease inhibitors when the target protein could be a protease that inte - (May/13/2008 )

Hello. It's me again. My problem this time concerns the use of protease inhibitors. My target proteins are PROTEASES and other proteins that interact with an endogenous inhibitor. If I add protease inhibitors to my lysis buffer, this might cause the dissociation of the endogenous inhibitor with some of my target proteins, and I will end up with non-protease proteins binding to the endogenous inhibitor. I Please advise. Thanks you.

since you don't want to add inhibitors then you need to work with conditions that minimize protease activity (especially if your protease autolyses).

we work at 0-4C at all steps. we also add edta to reversibly inhibit metalloproteases (but no other inhibitors).

if you know your protease and its inhibitors then you can add inhibitors for other classes of protease. if not then just work cold and quickly.

thanks for the suggestions. will discuss it with my supervisor before i start my work. thank you.