Hi all!
I'm looking for the immunodominant antigens of a pathogen recognized by the IgMs of the serum of some patients. To do it, I'd like to do an immunoprecipitation + mass spectrometry but I have certain doubts about how to do it.
IgMs do not bind well to protein A or G, so I have to use an anti-IgM. Should I do a premix of anti-IgM + beads and other of serum + cell extract and then mix everything? Would you have a proper protocol? Is it considered a co-IP?
Is there any problem with the pentameric structure of the IgMs?
Thank you in advance!!
Paloma.
Hello Paloma,
I'm not sure if I understood right, so correct me if wrong. You have beads coupled to anti-IgM antibodies and you plan to use them for fishing the IgM in sera that bound to any protein of the pathogen?
If it's that, then I'm following a somewhat similar protocol (but instead of anti-antibody beads I'm using Nickel coupled beads as my bait is a his-taged protein)
First you could mix the serum with the extract, mix gently at 4°C for a couple of hours. Then add the anti-IgM beads to the previous mixture and repeat the mixing. Finally separate the beads from the supernatant and wash them with a buffer so as any protein-protein interaction weaker than the antibody-antigen binding is minimized (as you would not like to identify proteins that interact with the antigens or the Fc but not with the Fab). I use Tris 20mM, 500 mM NaCl, some imidazole (between 5 and 10 mM) and 0,05% Tween-20 would also help. But any buffer you can find that is used for western blotting washes would work as well.
As for the naming of the procces, I guess it can be considered a co-IP, as your actual target is not the protein that interacts with the antibody in the resin beads (the IgM), so you'll co-precipitate both, the IgM and the antigens.
About the pentameric nature of the IgM I don't see any reason why it would interfere with the precipitation. At worst, you could get to the point where you get complex IgM-antigen-IgM webs that could let the complexes not-accessible to the beads, but it only happens after a certain ratio protein:antibody is reached, so be sure to use a lot more protein extract than serum so you avoid such troubles.
Hope it helps
You may use affinity ligand protamine that selectively binds IgM as protein A binds to IgG.
Thank you all!
Edward, yes, you are right. Thank you so much for the protocol. My starting idea was to couple covalently an IgG anti-IgM to the beads, mix the serum with the extract and then add the mix to the anti-IgM beads. I try to reduce the amount of antibody in the eluate, that's the reason why I plan to use a covalent bond. If I do it in that way, I would obtain IgM+antigens in the eluate. Is there a way that I can separate the antibodies and the antigens after the IP?
Shamsher and Md, that's a good point, thak you! Maybe the protamine has less specificity than an anti-IgM antibody? There is not much documentation about that. In this experiment it is very important to have the minimum amount of other antibodies in the eluate.
Thank you again and sorry to bother you!
Paloma.
This protocol of using anti-IgM antibody is ok but it has to be anti species. If human IgM, it has to be anti-human IgM.
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