Co-immunoprecipitation: how to increase amount of interacting protein?
Posted 02 April 2010 - 02:09 PM
So I've been working on co-IPs for a good while, and I have finally solved all problems except the most important. I am trying to find out if protein X interacts with protein Y by using an antibody to protein Y. They are all at endogenous levels in the cells. I can pull down LOTS of protein Y with its antibody, but there's only a tiny amount of protein X that comes with it out of a bunch in the whole cell lysate. Does anyone have suggestions as to how I might increase the amount of protein X in the IP?
I lyse cells with modified RIPA buffer (no SDS, 1% NP-40 substitute, protease inhibitor tablet from Roche, phosphatase inhibitors). I preclear for 1 hour and then add beads covalently linked to antibody for 2-3 hours. I elute by boiling with SDS loading buffer.
Can things like incubation time with the antibody-coated beads matter in this case? I'm already pulling down pretty much all of protein Y with its antibody in whole cell lysate. Would pH matter? Any other suggestions?
If this doesn't turn out, I have a couple other antibodies to try for this IP. A bunch of papers have been published with co-IP data for these 2 proteins in other cell types, and they all use different conditions, and some use HA or FLAG-tagged protein X.
Thanks a lot!
Posted 02 April 2010 - 03:31 PM
Posted 04 April 2010 - 06:51 AM
It might be that this interaction is just not very robust or has a low affinity and so you only get a small amount of protein X. The fact that other groups have over-expressed protein X with epitope tagging makes me think this is true. You will get a better interaction with over-expressed proteins. This being said, 1% NP-40 is a lot for an interaction buffer. I use 0.1%. How much salt does your buffer have? Some interactions require salt and maybe stabilized with higher salt while other interactions are broken. Certainly your incubation times can influence the amount of protein captured so it's worth going overnight. This will most likely increase background though. You can also try decreasing your washing stringency. pH can also affect interacting proteins but this will require much optimizing to determine. Additionally, you may want to try crosslinking the proteins before the IP. I've seen this done to capture very weak or transient interactions. I am also wondering if you have tried to IP protein X. Perhaps if you can IP a large amount of protein X, protein Y will be readily detectible. However, I know antibodies are finicky and you may not have reagents to allow for this. My boss frequently says that Co-IPs are one of the most technically challenging assays so hang in there and good luck!!
Forcing "optimal" interactions just seems wrong on so many levels doesn't it? I mean, the whole protocol of a co-IP starts off as an artifact by simply lysing your cells and having proteins encounter one another that might normally be sequestered from one another within the compartmentalized cell. Hope your control IPs are not pulling out "weak" amounts of protein X...you are doing IgG alone and beads alone right? Hopefully the interaction you're chasing has been shown by a couple of other methods as well as co-IP...and ultimately mapped. Epitope tagging and overexpression is fraught with ambiguities that need to be controlled for. Adding a tag to a protein can seriously change it's fold, half-life or interactor properties. Food for thought. Sometimes "weak" interactions are telling you something.
Posted 05 April 2010 - 02:03 PM
My lysis (and wash) buffer has 150mM NaCl. Going down to 0.1% NP-40 and letting it go overnight sound like a good things to try. I'll go back and check what kind of salt concentration these other papers used. I can't decrease the wash stringency because protein X likes to bind non-specifically to antibody controls and even beads alone sometimes! That's been one of my other major problems, but I seem to have taken care of that. I do also have antibody to protein X and also another one to protein Y, but one is super expensive and the other was a kind gift from another lab so I'd rather not use any more of them until I need to. Like many other labs, money is tight for us.
Yeah, I'm just looking for some biochemical evidence that these proteins interact or can interact. I'm also working on some overexpression and knock down studies and some fluorescence microscopy, so I'm hoping that eventually I'll have enough different types of evidence to say with some certainty that these proteins interact in this cell type. Perturbing expression levels in other cell types does seem to have an effect, so I'm hoping the same will be true for me!
Thanks for the advice and encouragment.
Posted 05 April 2010 - 04:10 PM