ECL immunodetection

LOGAN logan at msdos.montpellier.inra.fr
Wed Apr 20 08:17:44 EST 1994


Hello,
Last week I posted a message asking for help with the ECL system as I thought I 
had a problem with endogenous peroxidase activity in my plant tissue, thank you 
to those who sent advise. Unfortunately the H2O2 treatment had no effect, so I 
have done several control blots and discovered that the problem is actually that
the secondary antibody, Sigma's anti rabbit HRP will bind to a large number of 
plant proteins non-specifically, i.e. no primary antibody and lots of bands. I 
have tried reducing the secondary antibody concentration [down to 1:100,000] and
a different blocking agent cocktail, no success there, and I have contacted 
Amersham who have not heard from anyone else with a similar problem. However, 
when looking back through some postings from the newsgroup that I had kept I 
came across a message from Zhonggue Xiong who while answering another query 
happened to describe a problem with non-specific bands in plant protein 
extracts, sounded very familiar. So finally my question is, has anybody out 
there experienced a similar problem? I have asked Amersham about there secondary
antibody, which they believe would not have this problem, but as it is rather 
expensive before I splash out can anybody out there confirm that the Amersham 
antibody will not have this problem?


Thank you in advance for any advise/comments

Helen Logan
Logan at montpellier.inra.fr




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