1 reply to this topic

[doubtdoubt]

I do two-times elution by incubating beads/antibody/chromatin complexes at 65'C for 15 min and harvest the supernatants before decross-linking.
My question is, does this step seperate the antibody from chromatin? In another word, is the antibody still bound with chromatin in the supernatant or it is discarded with the beads?
I'm planning to do a pilot experiment for re-ChIP. Unfortunately two antibodies are both anti-mouse. In this case it is necessary to make sure no remaining 1st antibody after the 1st round elution. Not sure this will work. Anyone has any suggestions?

Edited by doubtdoubt, 15 July 2012 - 07:07 PM.

[KPDE]

It's expected with an IP that you will elute some of your antibody unless the antibody is crosslinked to your beads. If you've ever done a co-ip experiment and not been careful about the primary and secondary antibody you use in the western you find this out pretty quickly.  I wouldn't expect it to be much different here.