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Western blot of Gal4 BD

Frederik [iso-8859-1] Börnke ricky_boernke at gmx.net
Wed May 2 05:21:51 EST 2001



vuk schrieb:
> 
> I would appreciate if anyone shared a thought on registering the
> expressed Gal4- Binding Domain by Western blotting. I have used a
> monoclonal Gal4-BD antibody by Clontech and at best I get  unspecific
> signals all over, with perhaps a stronger band near 22 kD in positive
> control (but not entirely non-existant signal in the negative control,
> only weaker than the positive). The isolated proteins are from Y190
> strain transformed with Gal 4-BD  , and PGBT9 plasmid was used. People
> from Clontech claim that the promoter in this plasmid is too weak to get
> a sufficient ammount of my protein.
> Thanks,
> Vuk
> 
> savv at medizin.uni-leipzig.de
> 

Indeed,

the truncated ADH promoter of the pGBT-9 is to weak drive sufficient
expression for regular western blotting. However, there is a publication
about protein detection from yeast harboring pGBT (Langlands K and 
Prochownik EV (1997) A rapid method for the preparation of yeast
lysates that facilitates the immunodetection of proteins generated
by the yeast two-hybrid system. Analytical Biochemistry 249: 250-252).
Never tried it myself but looks ok.

Hope that helps
Ricky
 
*******************************************************************

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





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