co-immunoprecipitation - (Sep/19/2006 )
For the first question, you can add excess of a competitor peptide: you sintesyze the peptide that the Ab is supposed to bind and put it in eccess with your Ab-protein complex. This is what, as far as I know, most people use. The alternative is that you crosslink your Ab to the beads before the coIP.
The second step is difficult: I don't know how to separate the proteins from the complex, still keeping their functions. Do you need to do functional assays after that? or you just want to see who these proteins are?
salts will help you to get rid of ionic interactions.
it's what I use when I want to IP. I don't get too much of other bands.
But if you want to co-IP, you can get rid of antibodies (at least the heavy chain) by cross-linking, then you load on your gel and you separate your proteins, allowing you to identify the co-IPed proteins.
I don't understand what you want to do exactly.
it's what I use when I want to IP. I don't get too much of other bands.
But if you want to co-IP, you can get rid of antibodies (at least the heavy chain) by cross-linking, then you load on your gel and you separate your proteins, allowing you to identify the co-IPed proteins.
I don't understand what you want to do exactly.
to Missele:
I think i have explained my question very clearly. If you can't understand my mean by now,maybe you should read this whole posts carefully .Who i am looking for is an adept of doing co-ip.i will try your way in my experience.Thank you for your suggestion!
to others:
i am not sure that there are people encountering the similar problem like mine.Co-immunoprecipitation is not a new method but the applied field is so broad.In my protocol,i use it to deposit the complex of proteins but those are not my ultimate aim.What i want to get is the single protein.Then those complex need to be seperated .Maybe the seperated protein is a new molecule,so i don't want to this protein is destroyed especially its' structure.at least it would be integrated enough to be detected on the analysis of MS and sequencing of amino acid.
I think you should do your coIP, remove the complexes from the Ab as I suggested (or crosslink the Ab to the beads) and then do MS or 2D gel. Whenever you find a new interacting partner, you can test its activity in a separate experiment. I don't think you can do everything at once.
zhouwuchina,
No need to get shirty, people (i.e. Missele and everyone else) are just trying to help you.
I'm not sure this will be that easy to do. I was about to suggest boiling in laemeli buffer but I realised that this will leave you unable to do the 1st D of the 2D gel. I'm not sure that IP is the best way to get a pure fraction. It's always going to be a mixture of your protein when you salt it off , proteins binding to it (and the ab unless you crosslink it), although the ab-ag interaction should be stronger. If you have some idea of what you think is binding just perform a western and blot with specific antibodies.
If you have no idea, have you though about using an affinity column (using the ab or a GST-fusion protein of your protein of interest)? Then you could elute off under different salt concentrations, pH etc. Yeast 2-hybrid is also another way to pick up binding partners.
Ceri