Basic question about PMSF

ChenHA via methods%40net.bio.net (by hzhen At freeuk.com)
Sat Dec 16 12:05:17 EST 2006


On Sat, 16 Dec 2006 06:57:28 GMT, dk At no.email.thankstospam.net (DK)
wrote:

>In article <ra85o2d3u8qufkd3bvaqp9092hmh1q4jus At 4ax.com>, ChenHA <hzhen At freeuk.com> wrote:
>>On 13 Dec 2006 09:03:47 -0800, "Breslauer" <breslauer1981 At gmail.com>
>>wrote:
>
>I almost always observe minor film forming on the surface after
>PMSF addition but have not really seen precipitate. (Could it be 
>because I always stir the buffer while adding?)

Probably.  I normally just add it to my samples in  50 ml tube and
then swirl to mix, but  I don't always see precipitation. It may be
protein specific, but I haven't really thought about it.   I suspect
some initial precipitation event may cause proteins to further
precipitate if that protein is susceptible to precipitation.

>
>>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.
>
>Absolutely correct answer. I'd also note that PMSF is unstable in 
>aqueous solutions and therefore needs to be prepared fresh and in 
>solvents that contain least amount of water. I always assumed that's
>the reason for isopropanol. (?)
>

That's correct.  The reason for using organic solvents is because PMSF
is easily hydrolysed in water.  The half-life is something like 30-60
minutes in water (faster degradation at a higher pH  I think).  It
doesn't need to be prepared fresh since it is stable in isopropanol or
other organic solvents, or at least I haven't seen any problem with my
PMSF stored in isopropanol.


>DK



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