using IgM antibodies in IP - (Jun/19/2007 )

Hi

I have new antibody that I want to use in an IP, and I was just wondering if there are any special conditions that I need to know before I use the antibody- and has anyone ever worked with an IgM antibdy before??

Thanks, Neetha

You can use IgM for IP's two different ways. First is with Protein L. Protein L binds kappa light chains on antibodies, so the isotype doesn't matter, but you do need to have the right kappa light chains. This may not work for your antibody, but if it does, it's just like using Protein A or Protein G.

http://www.piercenet.com/Products/Browse.c...;WT.mc_id=forum

The other way is to take your purified IgM antibody and immobilize it covalently to a bead and use that for your IP. The nice thing with this method is that you don't get a lot of heavy and light chain eluted with your captured protein. Here is a kit that is based on that principle.

http://www.piercenet.com/Products/Browse.c...;WT.mc_id=forum

ok, but do u know anyone's who's used Protein L for IgM's? Or anyone who has an IgM monoclonal antibody? I've heard about Protein L, and also mannan binding proteins but there are no references that it will actually work in an IP.

I've seen work (unfortunately I don't have a reference handy) where protein L has been used for an IP with IgG - if it doesn't interfere with IgG binding, it will undoubtedly work with IgM which has 10 antigen binding sites. The same could be said for the covalent immobilization procedure.

I don't think I would use mannan binding protein because it's a large multimer and I think it's a bit unstable - likely to cause extra background.