co-ip: how to get rid of the ip-protein? - (Feb/17/2006 )
I am trying to pull down a certain protein to search for new protein binding partners within the cell.
The goal will be to identify new proteins via Mass Spectrometry.
My IP works nicely - I have a lot of protein in my pulldown. Using silver staining I also see additional bands that are not due to my pulldown method or my primary protein. However I need a lot of IP-material to see my additional bands. Thus I will likely contaminate my samples for MassSpec with my primary protein.
Is there a way to elute proteins that bind to my IP-protein without eluting the IP-protein itself?
The system I am using is the FLAG-epitope from Sigma. Currently I am eluting the IP-protein via a specific FLAG-peptide.
Thanks for your help in advance,
Hey great It sounds like we are doing very similar things! I just posted a question on the same topic.
I don't see why you don't want to elute your protein of interest. What I have been trying is eluting with just sample buffer, running on 1D gel, and then sending off to mass spec. I have been borrowing a large gel box (I think like 20cm) and running out my samples slowly and carefully on gradient gels. If your resolution is decent I don't think it should be a problem.
If the co-precipitating proteins are really faint in comparison to your protein of interest (it seems they always are) have you already optimized your buffer composition?? (ie amount of salts, detergent etc...)
Anyways, good luck
PS Is the FLAG epitope elution working well for you? How do you do it? does it interfere with the Mass Spectrometer? Did you consider/try using enterokinase to cleave the tag?