Removal of E.coli non-specific binding

Bernard Murray bernard at elsie.nci.nih.gov
Mon Dec 30 16:46:45 EST 1996


In article <01bbf68b$58a33a80$cd7106c2 at david.pulford.pop-3.ukonline.co.uk>, 
david.pulford at ukonline.co.uk says...
>
>I'm looking for a quick and easy way of removing E.coli antibodies from a
>rabbit serum. I've found pseudoscreening an incomplete, time consuming and
>expensive way of removing this background activity for screening an E.coli
>expression library. I chanced upon a method a while back that used
>formalised cells but have no reference for this approach. Can anyone help?

I don't know if this helps, but...
In a similar situation we found that the *second* antibody was the
major source of the "non-specific" reactivity in Western immunoblotting
of bacterial or yeast extracts.  By sampling a variety of second
antibodies (and dropping the amount used) we greatly reduced the
non-specific binding (we were using peroxidase labelled antibody).
We saw an even cleaner result when we switched from the second
antibody to HRP-labelled gammabind G so the extra binding may have
been through the Fab part of the immunoglobulin.
	Maybe try bacterial extract in the blocking buffer?

		Bernard
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)




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