co-immunoprecipitation help! - (Oct/12/2005 )
:( I'm from Brazil and I have one question about co-immunoprecipitation. I would like to know if I can compare the amount of ligant binding between two groups, after the elution of the immune complex, in SDS-page. Can I quantificate the proteins after the elution of the immune complex and do the SDS-page with same proteins quantity? In my case, I would like to know if the quantity of b-catenin binding to GSK-3 is different in two different cell treatment groups. I hope somebody could help me. Thank you
katiu, if you are using the same cell line - means they express your protein you pull down at the same level - then there is no problem. you just take, lets say, two different sets of different treated cells, lyse them, determine the protein concentration in your lysates. then you IP in the same conditions using equall amount of the imput lysate and using same amount of Ab (and the beads and so on), and then look for your interactor.
if you want to compare different cell lines with different expression, that's more painfull. i guess you'll have to optimalize a bit. for example, you could try to do an IP with equall amount of ab using an excess of a lysate from both cell lines, so you make sure you'll saturate the beads completelly, and IP same quantity. i tried that once, and it worked for me just fine...