What to do if the Solubilization of your Proteins is difficult?

Dr E. Buxbaum EB15 at le.ac.uk
Fri Dec 5 17:55:56 EST 1997


Martin Hartenstein wrote:
> 
> Hi,
> Can anyone working with difficult protein pellets help? I want to purify
> cellular proteins from an organic (phenol) phase, from which I have already
> dialyzed the phenol using 0.1% SDS. I think this was successful & I'm now
> concentrating up the dialyzate using Microcon concentrators; however, the
> dialyzate also contained a seemingly membranous precipitate which I assume
> to be proteinaceous (+ other) cellular material 

You can try to heat (or rather warm at 37 degrees for 60 min) in SDS
sample buffer, if necessary including 6 M Urea. This should unfold your
protein, as required for electrophoresis. Proteins will normally run
quite well in a Laemli system under these conditions. I know however of
at least one which doesn't (P-Glycoprotein). 

You may want to try different precipitation procedures, like
Chloroform/Methanol (Wessel & Fluegge). Recontitution seems to be
somewhat easier. 

In any case do not expect to get a properly folded, active proteins out
of such procedures. Electrophoresis is basically all you will be able to
do with them.



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