Help ! Problems with immunodetection on western blot

Nathalie Druart nathalie.druart at univ-lille1.fr
Tue Jul 8 02:58:22 EST 1997

Dear Netters,

	I have problems with immunodetection on western blot. I am working 
on chicory protein extract which contains 10% glycerol, 25 mM K phosphate 
buffer pH 7.5, 5uM FAD, 0.5 mM MgCl2, 50 uM TPP and 0.25 mM DTT. The 
extract have been tested positively for acetolactate synthase (ALS) 
activity. Six month ago, the antibody (rabbit) against ALS has been tested 
and was specific. And now, I have one big problem: with or without primary 
antibody, the secondary antibody (BIORAD, goat anti-rabbit alkaline 
phosphatase coupled one) detects the whole proteins as if it was a naphtol 
blue black coloration !
	What can I do ?
	Have you already had such a problem ?
	And did you resolve it ?

	I would appreciate any kind of advice or idea that could help me. 
Could you mail it or them  at: edewaele at pop.univ-lille1.fr

					Thank you, Delphine

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