[Histonet] Rabbit Anti-sera Background

[Bryan Hewlett]

Richard,

I'll bet that this rabbit antibody is an Ig fraction of whole serum.
As such, only up to about 10% of the available Ig's are directed against 
your antigen of interest.
The remainder are directed against assorted other antigens, some of which 
may be human.

1) Try switching your protein block from 1% BSA to non-immune goat serum ( 
to match the secondary),
2) increase primary antibody dilution to say 1: 5000-10000 or more, and 
incubation time to overnight @ 4C ( dilutes out other naturally occuring 
antibodies which are usually at a lower concentration than your primary),
3) add 10-20% pooled human serum to diluted primary ( to adsorb anything 
against human proteins).
If this doesn't work you may have to adsorb with tissue powders!

Bryan


----- Original Message ----- 
From: "Richard Bruggeman" <rbruggeman <@t> psu.edu>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Wednesday, August 17, 2005 10:30 AM
Subject: [Histonet] Rabbit Anti-sera Background


> We are having some problems with extensive background and nonspecific
> staining with a new rabbit polyclonal anti-sera.  Here are some of the
> staining details:
>
> -FFPE human tissue
> -1mM EDTA pH 8.0 HIER for 20 minutes in steamer seems to provide best
> staining
> -10% H2O2 block for 10 minutes @ RT (standard for our lab)
> -block with 1% BSA in TBS for approx. 30 minutes @ RT (no rinse
> afterwards, just drain off blocking reagents)
> -primary antibody dilution worked out to 1:1000-2000 (diluted in Dako
> diluent) for 30 minutes @ RT via titration runs
>
> -Dako Envision+ Goat Anti-Rabbit/HRP Labeled polymer detection used for
> 30 minutes @ RT (standard for our lab)
> -Dako DAB+ for 10 minutes @ RT (standard for our lab)
> -counter stained w/ Dako Hematoxylin for 1-2 minutes (standard for our
> lab)
> -All rinses done in  Dako TBS w/ 0.05% Dako Tween 20 (standard for our
> lab)
>
>
> We have tried doing peptide blocking experiments that have shown some
> of the staining can be blocked, however most of the staining is still
> present, suggesting that it is due to nonspecific binding.  We know from
> Westerns that our blocking experiments include a VERY large excess of
> peptide, so all of the specific antibodies should be bound to peptide.
> Negative controls (without antibody and/or with peptide only) always
> show up DEAD negative.  No avidin/biotin to worry about since this
> detections system is a labeled polymer.  The detection system has been
> working very well for us in the past, including with other commercially
> available rabbit polyclonals, so I can't imagine this would be the cause
> of our problem.  What suggestions might you have to reduce this intense
> background/nonspecific staining.
>
> Richard "Trey" Bruggeman
>
> Milton S. Hershey Medical Center
> Penn State College of Medicine
> Department of Pathology
> 500 University Drive
> Hershey, PA  17033
>
> (717) 531-1044
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