RE: Urgent question

From:"Connolly, Brett M" <>


Two ideas:

Since the rabbit IgG negative control is staining be sure you are matching
the IgG or protein concentrations of the rabbit antibody and the control IgG
when making your dilutions. Often the stock control IgG's are much more
concentrated than the antibodies. Then do a dilution series and go way out.

Second, get a rabbit anti-rat (mouse adsorbed) as Barb says, Jackson
Immunoresearch and KPL have good ones too ....same idea as Chris's idea of
preincubating with mouse serum.

Also, If it's a commercial antibody try a different source and/or species as
some are cleaner than others. Sometimes it helps to dilute the primary in
the blocking solution add Tween to buffers, use Dako's biotin-free Envision,

Importantly, don't forget to say prayers, wave magic wand, throw salt over
shoulder, chant your mantra and the like. :-)


Brett M. Connolly, Ph.D.
Merck Research Laboratories
Department of Pharmacology
PO Box 4
West Point, PA 19486
Ph. 215-652-2501
FAX 215-652-2075

> ----------
> From: 	Lynn Gardner[]
> Sent: 	Thursday, January 25, 2001 8:17 PM
> To:
> Subject: 	Urgent question
> Dear Friends,
> We are staining for anitbodies in nuede mice. They have been injected with
> rat cells and we want to demonstrate the antibody in the rat cells. The
> antibody is a rabbit anti-rat antibody. The problem is we get staining in
> the tissue but everything is staining. The more worrysome problem is that
> the Rabbit IgG negative control is also staining vibrantly positive even
> more so than the antibody stained tissue. Does this mean that the rabbit
> anti-rat antibody and the Rabbit IgG are cross reacting with the mouse
> tissue? or something else?
> I block for Hydrogen peroxide, avidin and biotin and use a protein block
> that is serum free. I can't figure this one out. Any hints would be most
> appreciated!!!
> Thanks gang,
> Lynn 

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