[Histonet] Peptide Competition

[Rebecca Jo]

Hello Histoneters-

I am doing peptide competitions (in tissue) with our antibody, but I've 
been getting some puzzling results.  I'm incubating primary antibody 
with specific or non-specific peptide (10X concentration of primary) 
overnight at 4C.  Then I spin them down and proceed with my usual 
staining protocol.  I also use a positive control where I use just 
primary antibody with no peptide which is also incubated overnight and 
spun down.  My problem is that I'm getting a pretty weak signal in both 
primary alone and primary + non-specific peptide.  Is it possible that 
my signal weakens because of the extra day of incubation (incubation 
with primary + peptide overnight and then incubation of primary/peptide 
with tissue overnight)?  If so, why would this be?  I'm asking this 
because when I stain with our antibody I get a very strong signal, but 
add that extra day of incubation for the peptide competition and my 
signal goes way down.  Any suggestions as to what might be going on 
here?

Thanks,

-- 
Rebecca E. Jo
UNC-School of Medicine
Department of Cell and Developmental Biology
(919)843-9648
CB# 7090