K+ SDS precipitation

Wolfgang Schechinger hubahopp at gmx.de
Tue Jan 6 16:04:10 EST 2004


Your proteins precipitate together with the KDS since the DS is attached to
the proteins and won't come off before precipitation. In fact, this feature
is used in minipreps when the SDS/NaOH solubilized bacterial proteins are
precipitated by adding potassium acetate.

So you'll need to get rid of the potassium ions. TCA should be fine for
precipitation. For solubilisation, use (SDS containing) sample buffer
buffer. If your proteins of interest really don't solubilize, dialyzation
is an alternative you might consider.

Wo

At 10:04 06.01.2004 -0800, redeamer wrote:
>Happy New Year to All!
>
>I have a problem:
>
>I ve prepared eukaryotic cell extract using a buffer, containing
>potassium (Hepes-KOH pH 7.9). I need this extract for western blots.
>As expected
>when i add protein loading buffer (SDS inside) all the stuff
>precipitates
>on entering the well of polyacrylamide gel :(.... I run the gel.. and
>had
>only part of desired signals on western (maybe because of material
>insufficiency)....
>
>However, does anybody knows wheter this precipitation alters the
>electrophoretic mobility of proteins or resists the entering of
>proteins
>into the gel? ... and how to get rid of K+ (phospate salts...) if I
>really
>need to do that...
>
>I can precipitate it with TCA, or? Actually i have already talked to
>one biochemist at our fascility and he told that TriChloroAcetic acid
>precipitation is a quantitative one and thats fine for me... hope no
>problems will arise with solubilization of my proteins after 80%
>acetone wash
>    =20
>thank you
>
>
>Drew
>
>
Wolfgang Schechinger


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