Hi there,

Do you run SDS/PAGE under reducing conditions?

If in non reducing condition, it might be that your protein got reduced during experiment and then its disufilde bonds got disrupted. The reduced form would run less than the native one on SDS/PAGE (because the former is less compacted than the  latter).

Another explanation is that your protein got modified during experiment (phosphorylation for instance). Is the interaction you are looking at involve a modigying enzyme like a kinase?

Is it possible that the pulled-down form of that protein had been modified? I mean post-translation modification. Maybe there are at least two form of the protein, modified and unmodified form. Just a little portion of modified form is in the crude extract. After co-ip, it enriched the modified form, so now you can see a clear band with slightly increased molecular weight.

However, that's just my assumption. How do you think?

Dominique Liger,

I performed SDS-PAGE  under reduced conditions...however i used same sample loading buffer for both crude extracts and co-ip complex extracts.. so, i feel there is no chance of changing molecular weight because of loading buffer. On other hand, i am trying to pull irf7 protein, which has differnt forms. As u said it would have gone modifications during experimental process. and It was reported in literature that increased shift of molecular weight  happening with few proteins after co-ip reaction but no where explanation was provided why it is happening so .

Yeah, I agree with Yu-Le Wu. You may be enriching your IP with post-translationally modified protein, possibly phosphorylated.

You can try treating your IPed product with alkaline phosphatase to see if the shift is gone.

Good luck.