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I have strategically placed myc epitopes into my protein of interest
and have found that, while the site is there by sequence, only some of
the sites are accessable to the monclonal antibody I am using to
recognize the epitope on a Western blot. The antibody does, however,
recognize the site by immunofluorescence and flow cytometry. Since I am
working with a membrane protein it is possible that it is not completely
denatured under the conditions I have used for electrophoresis. I have
tried excess SDS, 100 mM Dtt, 8 M Urea, incubations at 37oC or boiling
(in different combinations) with no success. I did get partial success
in denaturing the protein further by alkylating with iodoacetamide after
SDS and DTT treatment. I would like to try Guanidine HCL but have not
found a protocol that would be easy to use with several samples that
would need to be run by PAGE. Does anyone out there have any
suggestions?
Marge
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Content-Description: Card for Margery A. Connelly
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n:Connelly;Margery A.
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org:SUNY at Stony Brook;Dept. of Pharmacological Sciences
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email;internet:connelly at pharm.sunysb.edu
title:Research Assistant Professor
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fn:Margery A. Connelly
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