Rafael Garcia <rgarcia at errc.ars.usda.gov> wrote:
> I am solubilizing difficult proteins using a pretty harsh extraction solution:
> 7M Urea
> 2M Thiourea
> 0.05 M DTT
> 0.01M Tris
> 2.5% SDS
> 1% N-lauroylsarcosine (detergent)
> protease inbitor cocktail
> pH7.4
> Something in this mixture makes SDS-PAGE gels look streaky and dark.
Horrible mixture (typing errors?). A lot of things, particularly
denaturing salts and virtually no buffering (10 mM Tris??). Does a
standard SDS-PAGE really give you anyhing at all? (gel System?)
> If I clean up
(meaning what?)
> and concentrate the extracted protein with ultrafiltration, the gels work
> much better. (dialysis followed by lyophilization doesn't seem to work). Which component
> in this extraction solution is causing the problem?
Not enough info.
I would look at the Urea first. Surely there is a reason why you have
it there. But 7M? You are boiling things in SDS - 2-mercapto anyway, is
it that resistant to denaturation? (I know it happens, but these are
extreme conditions).
--
Kaj
