ECL immunodetection

Stacy Ferguson sferguso at kimbark.uchicago.edu
Sun Apr 24 13:34:07 EST 1994


In article <2p3a1o$hga at mserv1.dl.ac.uk> LOGAN <logan at msdos.montpellier.inra.fr> writes:
>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
>


Hi Helen,


I have had similar problems with the ECL kit, and it's a matter of working
out the conditions. Try several blocking agents. I've used BSA and Blotto.
Some batches of each work well and some don't. Get different lots of milk
and try them out.

Secondly, I have found that for my protein (non-plant and there's no logical
reason why background would be detected on plant blots and not others), I
can decrease my secondary antibody a great deal without compromising signal
quality. THe recommended dilution of secondary Ab is something like 1:1000
to 1:3000 if I recall correctly. When using that high a concentration, I 
get extremely dirty blots with lots of non-specific bands. At 1:10K to 1:30K,
my blots are text-book quality :) 

I would just titrate out reagents if I were you.


Good luck,

Stacy




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