Basic question about PMSF
(by ben.long At yourfinger.anu.edu.au)
Mon Dec 18 22:07:10 EST 2006
Dr Engelbert Buxbaum wrote:
> Breslauer wrote:
>> 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
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.
Remove "yourfinger" before replying
More information about the Methods