Best Secondary Reagents for Rabbit Polyclonal Abs

William H. Schott wschott at jax.org
Thu Jun 17 15:06:07 EST 2004


Please note this is JACKson Immunoresearch, not The Jackson 
Laboratory, we get calls all the time from confused people who try to 
buy secondary antibodies from us.
-will

>Hi,
>I use a biotinylated donkey anti-rabbit IgG from Jackson, in 
>combination with either streptavidin-PE or streptavidin-APC, and get 
>good staining for CCR8 on human cells. However, the primary rabbit 
>anti-CCR8 is affinity purified (ie, purified on the basis of binding 
>to the peptide used for immunisation), and I suspect that this makes 
>a BIG difference in our ability to get good staining. For a negative 
>control, I pre-block the anti-CCR8 primary with the immunising 
>peptide.
>
>For the detection of CXCR5, I have also used anti-rabbit reagents 
>from Sigma (F(ab')2 fragments conjugated to either FITC or PE) with 
>good results. Again, the primary antibody was affinity purified. But 
>in this case, I found that "ChromPure" rabbit IgG from Jackson was a 
>good negative control, as it gives a similar level of background 
>staining to the peptide-blocked antibody control.
>
>Good luck!
>Lisa
>
>At 11:40 6/16/2004 -0400, Hastings, Richard C wrote:
>>Hi,
>>
>>Lately, I have been facsing many rabbit polyclonal antibodies. I have been
>>using Molecular Probes goat anti-rabbit IgG Alexa Fluor 488 and Jackson
>>Immunoresearch donkey F(ab')2 anti-rabbit IgG (H+L) FITC or APC as secondary
>>reagents. I use fluorochromes that don't overlap with propidium iodide
>>because I like using PI as a marker of cell death (yeah, I know PI is more a
>>marker of membrane permeability, so humor me a bit here and I do know that
>>APC and PI overlap in their emission wavelengths). Problems I have run into
>>are these secondary reagents are not bright at all, the rabbit IgG preps
>>tend to have a lot of non-specific binding and purified rabbit IgG is a
>>lousy control.
>>
>>I have also tried the Molecular Probes rabbit Zenon kit and the results were
>>less than satisfactory. Anyone have any suggestions?
>>
>>Richard C. Hastings
>>Associate Scientist
>>Target Development/TBD
>>1800 Concord Pike
>>Wilmington, DE 19803
>>302-886-2452
>>richard.hastings at astrazeneca.com
>
>Lisa M Ebert, PhD
>Post-doctoral scientist
>Theodor Kocher Institute
>University of Bern
>PO Box 99
>CH-3000 Bern 9
>Switzerland
>phone +41 31 631 4160
>email lisa.ebert at tki.unibe.ch


-- 
William Schott
Flow Cytometrist
The Jackson Laboratory
600 Main St.
Bar Harbor, ME 04609
Phone: (207) 288-6192
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