Immunoprecipitation - is crosslinking neccesary? - (Jul/02/2006 )
OK, I am a bit pussled about this techniq. If I immunoprecipitate a protein, will it pull along interacting proteins as well (co-immunoprecipitation) or do I have to crosslink in order to see co-immunoprecipitation?
Will I be able to see co-immunoprecipitated proteins in a coomassiestained gel or is it to insensitive?
Thanks for the help.
I think it depends on the specific interaction, but I would say that you would have a harder time publishing an interaction if you can't see it without crosslinking, although it helps for making stuff easier to see.
As for a coomassie stained gel. Yes it is possible that you could see new bands in your IP's vs. control IP. I would recommend colloidal coomassie though as it is 10x more sensitive and should help you do this. The most sensitive is obviously western if you have a candidate protein though and the bucks for an antibody
Good Luck
I do coimmumnoprecipitations without crosslink. It is not a trivial technique and you have to find the right buffer condition for each coIP, varying in most of the cases the NaCl concentration. Let me know if you need a protocol
Hi, yes, I would very much like to have a protocol. I have done co-IP in 1% NP-40, 20 mM Tris, 150 mM NaCl and 1 mM MgCl2, but the only thing I see on coomassie is the protein my antibody is directed against. No co-precipitated bands despite the fact that I know that my proteins come together as a heterodimer on the cell surface.
Any hints are wellcome!
Hi Kenzo:
Are you sure that you are correctly extracting your two proteins as a dimer from the membrane, in a mild soluble extraction buffer? Maybe you´re only extracting a monomeric population of your target protein from the cell and the membrane-bound, heterodimeric one is not present in enough quantities to see the interaction with the other one (specially in a Coomasie gel; maybe you could try a WB anyway against the heterodimer partner?). I´m not very familiar with working with membrane proteins, but, is there any way to confirm that you´re extracting the membrane-bound population? Bear in mind that the composition of your buffer might be not strong enough? Is there any way to obtain an enriched fraction for your membrane related protein?
Miguelon
When I'm in th elab tomorrow, I'll send you the protocol and the recipe of th ebuffer I use to your PM
Are you sure that you are correctly extracting your two proteins as a dimer from the membrane, in a mild soluble extraction buffer? Maybe you´re only extracting a monomeric population of your target protein from the cell and the membrane-bound, heterodimeric one is not present in enough quantities to see the interaction with the other one (specially in a Coomasie gel; maybe you could try a WB anyway against the heterodimer partner?). I´m not very familiar with working with membrane proteins, but, is there any way to confirm that you´re extracting the membrane-bound population? Bear in mind that the composition of your buffer might be not strong enough? Is there any way to obtain an enriched fraction for your membrane related protein?
Miguelon
Hi Miguelon,
no, I can not be sure that I get the dimer. I tried WB with ab against the heterodimeric partner, and there comes up a band of approximately the right size. However, the ab is a very poor one and it crossreacts heavily with many other bands in the WB. At least I think that I get the heterodimeric partner together in the IP, but that it is only a very small fraction of it. And I expect it to be equal amounts (the heterodimeric partners come together already in the ER/Golgi and are not monomeric on the extracellular side).
How to prove that the membrane extraction works... and that I am not only extracting fragments of intact membrane... I have no idea... What about extracting in a very aggressive buffer containing SDS and also maybe Na-doc that should solve the membranes. Then maybe dialyse against PBS and do IP on that fraction. Would the heterodimer refold and come together again maybe?
Anyone who knows?
Hi Kenzo:
Are you sure that you are correctly extracting your two proteins as a dimer from the membrane, in a mild soluble extraction buffer? Maybe you´re only extracting a monomeric population of your target protein from the cell and the membrane-bound, heterodimeric one is not present in enough quantities to see the interaction with the other one (specially in a Coomasie gel; maybe you could try a WB anyway against the heterodimer partner?). I´m not very familiar with working with membrane proteins, but, is there any way to confirm that you´re extracting the membrane-bound population? Bear in mind that the composition of your buffer might be not strong enough? Is there any way to obtain an enriched fraction for your membrane related protein?
Miguelon
Hi Miguelon,
no, I can not be sure that I get the dimer. I tried WB with ab against the heterodimeric partner, and there comes up a band of approximately the right size. However, the ab is a very poor one and it crossreacts heavily with many other bands in the WB. At least I think that I get the heterodimeric partner together in the IP, but that it is only a very small fraction of it. And I expect it to be equal amounts (the heterodimeric partners come together already in the ER/Golgi and are not monomeric on the extracellular side).
How to prove that the membrane extraction works... and that I am not only extracting fragments of intact membrane... I have no idea... What about extracting in a very aggressive buffer containing SDS and also maybe Na-doc that should solve the membranes. Then maybe dialyse against PBS and do IP on that fraction. Would the heterodimer refold and come together again maybe?
Anyone who knows?
After you lyse the cells with th ebuffer for IP, spin down and get the pellet. Use 8M urea to dissolve the pellet and load it on the gel. Probe with the Ab to check if you have your proteins there