K+ SDS precipitation

EK nobody at elnino.com
Tue Jan 6 15:31:00 EST 2004


"redeamer" <aniko at ut.ee> wrote in message
news:115aa4f.0401061004.68d8a721 at posting.google.com...
> 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
>
> thank you
>
>
> Drew

Don't wash with acetone then and just resuspend in say 2x SDS loading
buffer. Alternatively, you could change the buffer in your samples by
dialysis or passing through a small gel filtration column, like NAP-5 from
Pharmacia.
Emir





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