I am trying to perform a co-ip using the µMACS™ GFP Isolation Kit but am so far having no luck. I am pulling down protein A that has a GFP-tag but when I probe for protein B I cannot detect it. I can see the GFP-tagged protein A when I probe the membrane with anti-GFP but I cannot see any signal when I probe with an antibody for protein B. These proteins DO interact and it has been published. I am not using the supplied lysis buffer, but a commercially made RIPA buffer from CST because it should preserve the protein interaction and this has been published. Any thoughts?
Hi Charles,
Since you did not mention what those protein A and protein B are, it will be difficult to guess the possibilities that could go wrong with your co-IP experiments. I have some general suggestions,
1. Before thinking in a complex way just run a straight western for protein B to make sure the protein is there and the antibody can see it.
2.Try the Co-IP experiment other way around. Pull down protein B and probe for protein A in western. Some antibodies work better for WB and some work better for IP.
3.Avoid using any SDS for washing the IPed conjugate (Protein of interest bound to antibody linked to protein A/G agarose beads). Any weak interaction between Protein A and protein B could be damaged by doing any harsh treatment with SDS in the washing step.
4.To dissociate IPed immune complex from the bead, use 5% β ME in the Laemli sample buffer and heat the samples at 70 deg C for 5-10 min.
5. Run SDS-PAGE at 4 deg C and transfer the proteins onto NCP/PVDF membrane also at 4 deg C.
6. Block the membrane overnight at 4 deg C with mild shaking instead of doing at room temp for 1h (Not sure what you actually did)
7. Try incubating with the specific primary antibody against the target protein for overnight at 4 deg C with mild shaking. You may increase loading quantity of the protein in the SDS-PAGE, increase primary/secondary antibody concentrations or try possible combinations to detect the signal .
8. Some proteins interact with each other at specific treatment and experimental conditions. Are you sure In resting cells these two proteins intearct? If not you have to modulate the experimental/treatment conditions as well.
Good luck!!!!
- Badges
- Science method