bernard at elsie.nci.nih.gov
Thu Aug 22 14:56:47 EST 1996
In article <Pine.SOL.3.91.960822141048.1166A-100000 at isnet>, smiles at bgsm.edu
>I'm doing a normal western blot, but our polyclonal antibodies seem to be
>binding to many of the E. Coli proteins. It is possible that the
>antibodies were raised to our protein, as well as any bacterial proteins
>present in the blood during an infection in the rabbit.
Just wondering, have you checked to see at what stage most of the bad binding
occurs? - is it there when probed with the secondary antibody alone?
The reason I ask is that we once had the same problem and traced
this to the secondary antibody (HRP labelled anti-rabbit IgG) and this
appeared to be the same with offerings from various manufacturers.
We switched to detection with HRP-labelled gammabind-G and much of the
background disappeared. I don't think that HRP gammabind G is available
any more - Sigma sell the biotin labelled protein (as protein G' -
catalogue number P8045) and Pharmacia sell it bound to Sepharose.
I suppose you could try protein G HRP but this tends to bind to albumin
as well as immunoglobulin. A more radical method would be to immobilise
some control e.coli extract and back-absorb the serum before use.
If it is indeed your primary antibody I'd suggest titrating it
down to see if that helps. This is especially necessary with
chemiluminescent detection systems.
If you find any magic blocking reagents I'd be interested to
hear about it.
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
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