ief gels

Sean Patterson seanpat at FMED2.UNCU.EDU.AR
Tue Dec 7 12:56:20 EST 1999


>Dear all,
>
>I am having considerable difficulty with separation of proteins on ief
>gels. Specifically, I am looking for a 100kDa integral membrane protein
>and a change in its phosphorylation state. Detection is by western
>analysis. I have tried all combinations of the following additions to
>increase the solubility of the protein in the gel and the loading
>buffer:
>
>2% Triton X-100
>2% NP-40
>2% CHAPS
>9.5M urea
>5% B-mercaptoethanol
>
>Best results in terms of intensity of bands on a stained gel are with a
>gel consisting of 4% acrylamide (37.5:1) and 5% ampholyte with the above
>additions less BME. Sample buffer is the same without acrylamide but
>including ampholytes and BME.
>
<snip>

It may not be relevant, but we had a problem with disappearing
immunoreactivity of a membrane protein on 2D's a while back. It turned out
to be the combination of urea and DTT - we got a partial loss of
immunoreactivity on Western with urea alone, but complete loss with the
combination of the two. We also originally thought the problem was the
protein not entering the IEF tube gel. You might want to try preparing your
sample with combinations of the two and running them on a 1D for Western
blot.

Hope this helps.



Sean Patterson, Ph.D.
Catedra de Fisiologia, CC33
Facultad de Ciencias Medicas
Universidad Nacional de Cuyo
Mendoza, Argentina
Tel: (0261) 420-5115 ext.2684/2747
Fax: (0261) 449-4117
e-mail: seanpat at fmed2.uncu.edu.ar





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