ECL optimisation

Glenn Cantor GCANTOR at VETMED.WSU.EDU
Mon Oct 9 12:31:17 EST 1995


Adam Boyt wrote that his Westerns with a biotinylated second 
antibody, which is then detected with strepavidin-HRP, was less 
sensitive than when he used a second antibody which was HRP-
conjugated, but not biotinylated.

One reason could be that he is using 10% milk as a blocker.  Milk 
contains some biotin, which would compete for the strepavidin-HRP, 
making detection less sensitive.  We had a similar problem with 
sensitivity, in detection of biotinylated immunoprecipitated 
proteins.  We switched blockers, and now use a casein preparation, 
sold by Pierce. Our sensitivity increased 5-10 fold.  

I'd be interested to know if this works for you, Adam.

Glenn Cantor
Dept Vet Microbiology & Pathology
Washington State University
Pullman, WA  99164-7040

gcantor at vetmed.wsu.edu



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