Basic question about PMSF

Bean Long via methods%40net.bio.net (by ben.long At yourfinger.anu.edu.au)
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 
86:574-579.

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.

-- 
Bean

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