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[Glycosci] Help in purifying unknown Drosophila membrane protein.

Partho Halder via glycosci%40net.bio.net (by partho.h from biozentrum.uni-wuerzburg.de)
Tue Nov 3 11:43:44 EST 2009


Dear Dr. Ericsson,
       
I am a graduate student at the dept of Genetics & Neurobiology, 
Wuerzburg Biocenter. I am working under Prof. Erich Buchner. I am trying 
to purify and identify an unknown protein from Drosophila (10 kDa in 
size) against which we have a monoclonal antibody, which is a one among 
the Wuerzburg Hybridoma Library antibodies. It has not been possible to 
pull down my protein by usual IP protocols, and then I found that when I 
homogenize heads in a detergent free buffer and spin homogenate at 
100000xg my protein remains in the membrane pellet. Cloud point 
precipitation of the membrane pellet dissolved in 2% TritonX114, showed 
that my protein remains exclusively in the detergent phase. Hence we 
believe it to be a membrane protein. As a result 2DE attempts (using 7cm 
stip Zoom IEF-PAGE from Invitrogen) also failed. In the mean time, we 
found interesting staining pattern of the antibody in specific layers of 
the medulla, lobula and lobula plate, whole ganglion (SOG & thoraic). It 
is also present in the posterior most cell of the oogonium (egg cell) 
and so gives strong western blot signal with embryos both within female 
abdomens and after being laid as eggs. The protein is also detected in 
these parts by immunostaining of cryosections. And some life stages like 
larvae show a second higher (just above 10kDa) band in westerns. We are 
very eager to study this protein which has such a small size, but 
widespread but specific distribution and so early in life cycle.

So in this regard I would like to request you to help us in purifying 
this protein (2DE or chromatography followed by MS or whatever you feel 
is suitable). We can send our monoclonal antibody or if needed I can try 
to come over, whichever is most convenient to you.

Looking forward to your kind reply.

Warm regards
Partho Halder

--------------------------------------------------
Graduate School of Life Science
Dept of Genetics & Neurobiology, Buchner Group
Labor DK39, Biocenter Am Hubland,
University of Wuerzburg
D 97074 Wuerzburg, Germany.
Phone 0049 931 3188045 (Lab)
          0049 176 24889399 (Mobile)



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