[ccp4bb] Protease inhibitor cocktails and protein crystallization.

[Pascal Egea]

Dear All,

I apologize if the questions has already been asked on this forum.
We are purifying a membrane that seems prone to proteolysis. Although we use
Protease Inhibitor cocktails during lysis and the first step of purification
we get rid of them after and only keep PMSF and EDTA as general
anti-protease control agents.
I am considering reincluding the cocktail of inhibitors at the last
purification step (a size exclusion in our case) and was wondering if having
this infamous mixture of peptidic inhibitors (for the most part) around
during crystallization would be a problem: specifically getting crystals of
these inhibitors.
Does anyone have extended experience in this matter.

Many thanks in advance.

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu

Re: [ccp4bb] Protease inhibitor cocktails and protein crystallization

[Mark A Saper]

Regarding adding inhibitors. I'm not sure the effect on crystallization but if 
you use too much you will get covalent modification of your protein from one of 
them (I can check my notes as to which one).  We searched many MS databases of 
protein modifications to realize why our protein was the wrong Mr. Then my 
student happened to mention that he had added 10 times top much. Well at least 
we now know what not to do. 

Mark Saper
sa...@umich.edu