I am starting an IP experiment.
Here it is.
I want to check interaction betweein human protein X and Human protein Y.
I did IP the ProtX with mouse AntiX, and proein A bead.Then I did my western with the mouse antiX as a control and mouse anti Y (first antibody) to check if I have interaction..
Ifinally I use an antimouse HRP (2nd antibody) for ECL detection.
I have a few question.
Concerning the Heavy and light chain, what antibody are they coming from? are they coming from the antiboy I use to ip (mouse antiX) or the first antiboy( mouse anti Y).
what is the bet way to get read of the HC and Lc. I heard to pick difference species of antibodies what is that?
Anothe matter: if Y and X interact , will the antiX able to puldown X since Xinteract with Y so the epitope will not be available.