No specific band in GAL4 Western

[Frederik Boernke]

Hi!

I am currently trying to detect the GAL4 fusion protein for my 2hybrid
screen. Therefor I use a Clontech monoclonal mouse Ab and Pierce
Super Signal Ultra Chemiluminescent Substrate
together with an Amersham biotin/streptavidin system.
The samples were prepared after the method of Printen and Spraque
(1994) and 20 ul were loaded onto a 15% mini gel. The primary Ab was
dilutet 1 : 10000, secondary 1: 100000 and the strepavidin-HRP also
1 : 100000. When I develop the blot the only thing I see is one major
band at about 50 - 50 kDa ( time of exposure: 10 sec). My question now
is, at which step should I try some optimization. Less Protein or higher
dilutions of Ab's. 

Thanks in Advance,
Ricky


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Frederik Boernke
Research Group Molecular Plant Physiologie
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel.  039482 -5 321
Fax. 039482 -5 515
e-mail: boernke at ipk-gatersleben.de
http://www.ipk-gatersleben.de