GPCR western blots

[leurs]

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
using: 

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

Advices are welcome!!! 

Greatings 
-- 
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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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