IP first-timer - doubts on equal protein concentration control (Nov/01/2007 )
I am trying to set up an IP experiment to study the association between two proteins and have quite a few doubts… If I immunoprecipitate a protein which varies in quantity with treatment conditions, and then I go look for the presumably associated protein in WB, how do I check for equal loading? I mean, is there any way I can make sure that IP efficiency was the same for all samples? Does it make any sense to do the experiment this way or should I just try a different approach?
Thanks for help & suggestions !
If you expect a black and white answer (association or not) you don't need to control equal loading, but usually it's not black and white, and you want to see an increase or decrease of the association. So, you should do a control western-blot where you show the amount of the immunoprecipitated protein.
Let's say you IP the protein A and you want to see the association with protein B.
do a western-blot with anti-protein B, quantify with optiquant or anything else, strip the membrane and western-blot with anti-protein A, quantify, and calculate a ratio to show increase or decrease of the association.
You're right!!! Many thanks, I'll do it the way you suggest!