GPCR western blots

leurs hoffmann at
Tue Oct 21 06:57:20 EST 1997

Hi there! 

I would like to know your experience in the detection of tagged 7TMR
(mainly HA, 
Flag) in WB. 
I have been working for a couple of months on this and had a lot of
problems with the 
detection (basically I always get a very strong background in both
transfected and 
non-transfected cells BUT NO specific bands). This is what I have been

Receptor expressed in COS7 cells at about 2-3 pmol/mg in binding assays
solubilized with different detergent mixtures (Triton, NP-40, SDS,
1st Abs from Boehringer and Kodak (HA and Flag respectively), tried
concentrations and different incubation times 
2nd Ab peroxidase conjugated 
ECL detection 

Advices are welcome!!! 

Marcel Hoffmann Ph.D.
Dept. of Pharmacochemistry 
Vrije Universiteit Amsterdam
de Boelelaan 1083, 1081 HV Amsterdam 
tel: +31(0)20-4447572
fax +31(0)20-4447610 

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