how to pulldown membrane-bound protein? - co-immune precipitation (Oct/06/2008 )
Hello everyone. I now encountered a difficult problem here.
I suspect one of the protein I work with interact directly with a membrane-bound protein and my boss asked me to prove this. I already have the fluorescent colocalization in vivo but she think it is not good enough and I should at least have a co-IP result or other biochem result. The protein I work with is a cytosolic protein but its interacting partner is a membrane bound protein, and I wonder if I can use a regular co-IP with it?
By the way, I have already considered using membrane raft pulldown but it is not an acceptable method on this particular case because the protein I work with has been shown to interact with another (different) membrane-bound protein. So if I just break down the membrane to lipid vesicles it will contain BOTH membrane bound proteins and I won't be able to distinguish exactly which one pull down my protein with.
Thanks in advance!
hey there...in my understanding of your problem, i somehow think that immunoprecipitation would be more applicable here...ifthe protein in colocalizing with another protein then you can see the expression by western blotting using antibody agunst your original protein...then immunoprecipitation with respectable antibodies should give you the result...also you might have to work with different concentrations of SDS for immunoprecipitation to optimize your receptor expression...