1 hr pre-incubation at RT should be sufficient. try performing with agitation to reduce precipitation of IgG-antigen complexes. I'm assuming your peptide is the antigen used to raise the antibody? How large is the peptide? You could try conjugating the peptide to a soluble carrier protein (BSA or KLH)

Try a faster or longer centrifugation to remove insoluble complexes.

Perhaps your (unbound/excess) peptide is just very sticky and is the source of the problem?

To get pre-absorbed ab from a monoclonal sounds strange. They do not form precipitate usually as polyclonals.You can use the peptide in high excess as competitor mightbe during the first reaction (i think this is the normal way in this case, but i am not sure).

Thanks a lot for your helpful answers!

Stephen -- yes, the peptide is the immunizing antigen for the antibody, and it is quite small. I think it's a good idea to try a longer/faster centrifugation, and increased agitation during the incubation.

Also, it may very well be that the unbound peptide is just sticky. I'm going to run a condition incubated with peptide only (no Ab) and see if I get similarly dark background.

Attila -- I think you're right that it's less common to do preabsorption with monoclonals. Poor/weird precipitation may be an issue. So far after the peptide-Ab reaction I've not observed an obvious pellet after centrifugation.

I may try to absorb the precipitate (and excess peptide) out via other methods, maybe incubating with a tissue from a different species or one that doesn't express my protein of interest.

I have experienced the same problem with preabsorption of antibodies during antibody validation procedures and have found the following methods helpful for alleviating the problem.  When using a polyclonal, I titrate the peptide concentration for optimal formation of immune complexes, which then can be removed by centrifugation.  However, as Attila says, that doesn't work with a monoclonal antibody.  Therefore, for monoclonals, I have coupled the antigenic peptide to sepharose and used affinity chromatography for preabsorbing the antibody. HiTrap NHS-Activated HP columns work well for this purpose.  This eliminates the problem of peptide sticking to the tissue sections and causing dark staining.  

Thanks again for more helpful answers.

John -- That's a good point. There is a KO line available as well as positive control tissue. The best course of action may be to avoid the preabsorption altogether.

Lynn -- That is extremely useful information, thank you!