mercaptoethanol ghost band in Western

Ricardo Silva Ricardo.Silva at CIGB.EDU.CU
Mon Sep 7 09:23:48 EST 1998


Question
Our group is working about G-protein coupled prostaglandin rceptors 
and our aim is to detect receptor proteins by western-blotting using
polyclonal rabbit antisera against the receptors. Unfortunatly, if we
reduce our protein samples to denaturate the receptors we see a very
prominant band with a molecular weight around 67 kd after treatment of
our blots with rabbit antisera wich is not vissible if we do not reduce
our samples. 
I would like to ask if someone have also observed this phenomenon and
have overcome this problem?
Thank you very much.
 
Dr. Frank Neuschaefer-Rube
Dept. of Biochemistry
Georg-August-University, Goettingen, Germany
Email: fneusch at gwdg.de
Tel: xx49 551 39 5950 / 5978 Fax: /5960
------------------------------------------

Possible solution:

We sometimes have detected the same problem with reduced 
protein samples, although we work on bacteria. It has to do something 
with the quality of the mercaptoethanol ("chemical aging"). To 
avoid this problem, we put mercaptoethanol at  -20 C for an hour or 
so, and then try to recover the less viscous fraction, leaving behind 
the floculated portion that will become evident if mercaptoethanol is 
indeed the problem (too old, usually). Then we use it as normally 
in the sample buffer for SDS PAGE or any other. 
Hope this will help,

ORLANDO LUIS PARDO LAZO
DIV VACUNAS, CIGB,
C. HABANA, CUBA. 






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