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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.

-yiklim-

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

-pumuki-

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.

-Doc_Martin-

QUOTE (Doc_Martin @ Oct 2 2006, 08:05 AM)
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

-MisticMatt-

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?

-yiklim-

QUOTE (yiklim @ Oct 3 2006, 02:05 PM)
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?


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.

-The Bearer-

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.

-pumuki-