Hi Roberto,

There are a number of strategies you can try, which I'll describe below.

1) You mentioned you are able to run your IP's on Western. Have you tried loading your IP's and performing silver stain to see what the gel quality looks like? You could submit individual bands that correspond to your bait and specific interacting partners (i.e. bands not present in your negative control) for identification.

2) One potential problem is excess of IgG eluted off the beads. Running your IP's on a gel could help you eliminate the IgG heavy and light chain bands by avoiding those areas when you cut out bands. Another strategy is to biotinylate the IgG antibody raised against your bait prior to IP. See this paper for a description ( https://www.ncbi.nlm.nih.gov/pubmed/?term=23295261). The goal is that the biotinylated antibody will still IP your bait (which you can test by performing a Western to see if a band is still detectable, understanding that even if your antibody detects your bait doesn't mean it can pulldown your bait but that if your antibody no longer detects your bait, it's unlikely it'll work for pulldown) and the biotinylated antibody can still be bound by protein-A beads. So when you elute your TF complex from the protein-A beads, you can clear the elution/flowthrough with streptavidin beads to remove excess IgG bands. The resulting flow-through from the streptavidin clearing step may improve the ability to detect your bait and interacting partners in solution.

3) How do you generate your nuclear preps? It's possible you're enriching for your TF that's not bound to chromatin (i.e. in nucleoplasm), which may or may not be appropriate for your goals. If you want to enrich for nucleoplasmic TF, a low salt extraction (100-200uM NaCl) should avoid disrupting nucleosomes and minimize histones in your input. It's unlikely that histones themselves would "obscure" your TF bait because histones are highly digested under standard proteomic workflows (i.e. trypsin based); the resulting peptides are too small and should not be retained on most C18-based columns.

Thanks,

Barry

I'd like to re-phrase what Barry wrote in other words:

IP for WB is not IP for proteomics.

WB is insensitive to the amount of other proteins in the sample - if your protein is there and your antibody works against it, the protein will be detected.

proteomics is super sensitive to the relative quantity of proteins. that means that if your sample is full of antibody (if you've used Protein A/G for IP) or at least light chains (if your mAb is directly coupled to beads), in quantities far surpassing the amount of any other protein.

then there is non-specific binding, which is not a concern to WB but it is for proteomics.

In our facility we encourage our clients to run a method-development step for their IP directed specifically for proteomics. we discourage the use of mAb-Protein A/G combination (or His-tag).

to give you specific advice I will need more details of your experiment - how you preform your IP, with what tag/antibody, you washes, elution protocol and QC procedures.

Cheers,

D.

We use RIME method. Check out this paper:

Article Rapid immunoprecipitation mass spectrometry of endogenous pr...

Thank you for the tips. We will try a few of your recommendations. David, I might reach out by email if necessary.

Roberto J Botelho sure, good luck!