No specific band in GAL4 Western

[Joe Boutell]

Hi Frederik

One thing to be careful of is that last time i looked the Clontech 
monoclonal GAL4 Ab didn't detect the fusion proteins produced from their 
original 2 hybrid system, only the higher expression system 2. However, 
things might have changed, and it sounds like you're using a more 
sensitive chemiluminescent system anyway (and you might be using the 
higher expression system). In which case, the one thing i'd say is you 
seem to be diluting your antibodies an awful lot - i tend to use 1:1000 
for my primary and 1:10000 for the secondary.
Cheers,
Joe Boutell



boernke at IPK-Gatersleben.de (Frederik Boernke) wrote:
>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
>
>
>******************************************************************
>
>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
>