Basic question about PMSF

Bean Long via (by ben.long At
Mon Dec 18 22:07:10 EST 2006

Dr Engelbert Buxbaum wrote:
> Breslauer wrote:
>> Listen,
>> is it normal that when adding PMSF (250mM solution in MeOH) to cell
>> free extract from E.coli (broked by sonication and spinned 12000g for
>> 30min) to the final concentration of 0,5mM buffer becomes cloudly and
>> st become to precipitate (probably protein?).
> No. At this concentration PMSF should not precipitate proteins, nor
> should it precipitate itself (provided that excessively high local
> concentrations are avoided during mixing). 

I was surprised to hear you had a stock of 250 mM.  I didn't think PMSF, 
even in methanol, was that soluble.  I usually make up a 170 mM stock in 
isopropanol.  Having said that, I'm sure that wouldn't be the problem. 
You may want to check one of the original reports on using PMSF as a 
protease inhibitor:

James, G. T. 1978. Inactivation of the protease inhibitor 
phenylmethylsulfonyl fluoride in buffers. Analytical Biochemistry 

You might find some clues here.

My first thought was that the methanol was the problem but the final 
concentration is too low to really be a problem unless you have 
extremely high [protein] and relatively little mixing upon PMSF addition.

Hope this helps.


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