Simulated monolclonals?

Bernard Murray bernard at elsie.nci.nih.gov
Sun Jun 15 14:35:02 EST 1997


In article <33A29ED1.4F4E at mother.com>, mikel51 at mother.com says...
>
>I have raised several antisera to E. coli produced proteins that give
>less than satisfactory results for Western analysis.  There is a
>relatively high level of background bands when using the antibodies
>against E. coli extracts.  I tried affinity purifying the antibodies
>against the his tag purified proteins used to inoculate the bunnies, but
>this didn't make a significant improvement.  My next thought was to have
>some peptide(s) synthesized, link to CnBr sepharose, and affinity purify
>antibodies that react with specific a specific peptide or peptides.  Can
>anybody give me some advice about whether this approach is worth
>trying?  Any suggested protocols?  Any advice about how to pick the
>peptides?  Any suggestions as to a good source of synthetic peptides?
>
>Thanks in advance,
>Mike

Our finding is that one of the major problems with blotting e.coli
extracts (or yeast extracts) is "non-specific" binding of the second
antibody.  It may be worth checking this and trying a few different
antibodies.  We found that HRP coupled to gammabind-G gave the
cleanest signal (better than a secondary antibody).
	Preabsorbing your antibodies (primary and secondary) with
negative extracts may also be a way to go.
		Bernard

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
(but soon moving to San Francisco)




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