Hi guys, I need to troubleshoot my IP for low protein in my elutions. I'm performing an IP experiment for a 6X his-tagged protein using an ChIP grade anti-his antibody and dynabeads, I saw overexpressed protein in the cell lysate (as shown in figure). However, it seems like my protein is not binding to the abntibody-beads complex at all. I'm using 100ul dynabeads A and 10ul of my rabbit anti-His antibody. My lysis buffer composition is Tris pH7.5 50mM, EDTA 1mM, NaCl 150mM, and 1% Triton. I incubated my antibody with beads for ~1hr first, then add my cell lysate for a 2hr 4C incubation. I wonder what's the amount of protein that they can see on the gel and any suggestions to get my protein binding to the antibody? This is a ChIP grade antibody and should be working and I tested its functionality with western blot as well.
Essentially nothing is binding to the anti-His antibody. Virtually all of the protein is in the unbound fraction. Many anti-His antibodies don't work very well. Instead of using an anti-His antibody, use Ni-NTA beads for the procedure, since you know that they bind your protein well, or an antibody that is specific for your protein rather than the His tag.
Adam B Shapiro Thanks for the suggestion! Yes, Ni-NTA column did give my very high yield of protein. However, I'll eventually do a chromatin immunoprecipitation and I haven't seen people doing immunoprecipitation with Ni-NTA. Maybe it's the EDTA in my buffer that interfer with Ni column?
Adam B Shapiro Unfortunately no currently commercial antibody is available for our protein. That's why I'm using a tag. Wonder what how thick the band should be in the elution with 100ul magnetic beads and 10ul antibody
Juechun Tang I tried IP with triple Flag-tag in C-terminal of proteins, I also observed it hard to see the bands in coomassie blue stained -SDS gel. You can try staining by silver or sypro Ruby.
Thuy Nguyen I just wonder what's the normal amount of protein that should be pulled down and how much should be seen in the gel. The only pic I found online seems like there's a great enrichment in the elution, but they didn't post the details. Do you do IP regularly? How much cell lysate do you load and do overexpress your protein (as I did?) so that there's a real thick band?
I expressed protein from hela cell line, 15cm dish for each, lysis in 200ul lysis bf and sonication for 30min, IP overnight and elutute buy flag peptide.
Hi Medhi, are you incubating your antibody with a whole cell lysate or just with the chromatin fraction?
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