Best Secondary Reagents for Rabbit Polyclonal Abs

Lisa Ebert lisa.ebert at tki.unibe.ch
Thu Jun 17 02:42:43 EST 2004


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




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