I am using immunoprecipitation to determine whether two overexpressed proteins interact. In the image below, the red band is the one that I have IP'd. There doesn't appear to be an interaction, but the IP'd protein has shifted up about 10kD. I can't figure out why this is. I am eluting the IP by boiling in SDS, and I have tried increasing the SDS concentration to 2%, thinking that it might be antibody fragments that are stuck on the protein, but this does not change anything. I am crosslinking the anti body to the beads so there is no IgG in the eluted sample. The same thing happens to the green protein when I IP it, and the red protein remains at its predicted size in the sup fraction. Any ideas?
In this experiment, the beads were boiled directly in 2x laemmli buffer (containing 2% SDS). The input and sup samples were mixed 1:1 with 4x laemmli buffer to keep the detergent concentration the same as the pellet fraction. They were then run on a 10% denaturing gel.
Thanks Christina, but in this experiment the antibody is crosslinked to the beads, so there is no IgG in any of the lanes on this gel. Furthermore, the green band is present in the input sample, which is before I have even added the antibody, and it is absent in the eluted sample. My question is simply why has the red band shifted up 10kD after the IP?
hi there, I just wondered if your antibody is entirely specific? Could it be bringing down a similar member of the same protein family? Also could the SDS be denaturing the protein enough to keep the peptide chain intact (ie you still only have one band on the gel) but that it slows down its movement in the gel? Perhaps another elution method may be more appropriate for your protein.
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