substitution for PMSF? - (Oct/02/2006 )
I have harvested my bacterial cells in pellet form. Now i want to resuspend them before gently lysing them to determine the solubility of the recombinant protein of interest. The resuspension buffer suggested is 2mM PMSF in PBS.
The problem is i don't have PMSF. What are the substitutes and their working concentrations? I was told to use DTT.
I use the following protease inhibitors:
leupeptin at 10 ug/mL
soybean trypsin inhibitor at 10 ug/mL
aprotinin 2 ug/mL
Also I use 1mM DTT.
I also strongly recommend CompleteMini tablets from Roche, they contain several protease inhibitors, work well and are very easy to use.
good luck
The most similar substitute for PMSF is AEBSF (from Sigma and others) which also contains a sulphonylfluoride group but is highly sluble in water and not as toxic but has high protease inhibition activity.
Prefabloc is another substitute.
I would look into complete tablets from Roche. Our lab uses them with good success (x-ray crystallography group, btw). Generally we use 5 mM beta-mercaptoethanol and complete tablets. Beta-mercaptoethanol is much better with metal affinity columns than DTT.
-Matt
Thanks everyone for your replies.
Can i use 5mM beta-mercaptoethanol alone as a substitute for PMSF? After all, it's a reducing agent like DTT.
I was suggested to use 1mM EDTA instead of PMSF by a researcher working on expression work. Has anyone of you had experience on using EDTA to inhibit proteases? Does it work?
Can i use 5mM beta-mercaptoethanol alone as a substitute for PMSF? After all, it's a reducing agent like DTT.
I was suggested to use 1mM EDTA instead of PMSF by a researcher working on expression work. Has anyone of you had experience on using EDTA to inhibit proteases? Does it work?
I think there is still a misunderstanding: PMSF is not used as reducing agent but inhibitor of serine proteases. Its sulfonyl group is used to sulfonize the catalytic site of serine proteases.
High concentrations (> 1mM) of reducing agent may inactivate several other proteins by cracking disulfid-bridges, therefore, it is not recommended; by the way, usage of mercaptoethanol might not increase your popularity in your lab...
EDTA and EGTA are used to inhibit metalloproteinases by complexing Mg2+, Zn2+, and Ca2+; respectively. It is absolutely recommended to use them in your protease inhibitor cocktail.
I agree to use Roche Complete tablets.
Well I really don´t know if EDTA can work as a protease inhibitor, but in my protocol (who somebody gave me once, I don´t remember) there is, apart from the compounds I post above, 1mM EDTA.