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

[Martin Hartenstein]

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 denatured and solubilized
in the presence of phenol, but which remains insoluble in its absence due
to its inability to return to a native conformation in the presence of 0.1%
SDS. Since I would also like to solubilize this in order to pool all of the
material together in Laemmli loading buffer for subsequent SDS-PAGE, does
anyone know of a protocol to carry this out? Presumably the proteins at
least can be solubilized in strong denaturants such as guanidinium or 8M
urea, but I guess the concentration of these agents should then be
gradually reduced in steps to prevent re-precipitation of the problem
proteins. Please, any comments?

Please mail to:
Martin.Hartenstein at bio-geo.uni-karlsruhe.de
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**  Dipl. Geooek. Martin Hartenstein                             
**  Institut fuer Geographie und Geooekologie I   
**  Universitaet Karlsruhe                  
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**  Tel.: +49 721 608 4722       Fax.: +49 721 696761 
** 
**  EMail: Martin.Hartenstein at bio-geo.uni-karlsruhe.de 
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