band shift after IP - (Jun/11/2012 )
I am using immunoprecipitation to determine whether two overexpressed proteins interact. One is fused to S-tag, the other is fused to mCherry. Irrespective of the fact that there is no evidence of an interaction in the experiment, I am still able to IP the individual proteins themselves. However, both target proteins, when IP'd with their respective antibodies, appear shifted up about 10kD compared to input and sup samples. 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. Any ideas?
Perhaps the Abs only bind to post-translationally modified versions of the proteins (e.g. ubiquitylated).