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.
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