Basic question about PMSF

ChenHA via methods%40net.bio.net (by hzhen At freeuk.com)
Fri Dec 15 08:43:28 EST 2006


On 13 Dec 2006 09:03:47 -0800, "Breslauer" <breslauer1981 At gmail.com>
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?).
>
>Thans for any clue
>

Be aware of silly answer given in newsgroup.  PMSF is not an alkylator
nor can it be described as non-discriminate.

We often use PMSF to inhibit serine proteases.  We normally make 100
mM stock in isopropanol,and used 100 to 1000X dilution.  Other
solvents apart from methanol and isopronanol  are ethanol and DMSO.
Occasionally we see some precipitation when it is first added, but
that quickly disappeared when mixed.   I always assume that the
preciptation is PMSF, because it is not very soluble in water, but I'm
not sure.  You should check what is precipitated, it should be easy to
tell with a gel if it is your protein.  Check also if it is the
solvent that is precipitating your protein if it is indeed the protein
that is precipitating.



>Julio



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