HELP: PROTEIN SOLUBILIZATION

[Ding Ming]

In article <36btke$jct at emory.mathcs.emory.edu>, medtjm at bimcore.emory.edu wrote:
> I am studying an integral plasma membrane protein (very hydrophobic)
that we suspect
> is forming an insoluble multimeric complex when prepared for SDS-PAGE
> (sample loading buffer=4%SDS, 2-ME, pH 6.8).  The results are the same
whether or
> not the samples are boiled.  Immunoreactivity on Westerns appears
> to be retained in the wells of the stacking gel. 
> Does anybody have any specific protocols for treating a plasma membrane
> fraction sample to overcome this problem?  

You may try add urea into the sample loading buffer, and heat the sample
at 50°C or so rather than boil it.

May I ask why you need to do the sds-page? If you want to know some info
about its MW, you can do a simple ESI-MS, in which a variety of solvents
can be used to dissolve the protein. If you really want to check the
immunoreactivity, a dot blot titration definitely works better in your
case.

+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
Ding Ming
University of Wisconsin-Madison
Telephone: (608)265-3544     Fax: (608)262-7420
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++