Dear Owen, basically, only an experiment will tell you if it works. I'd carefully check the conditions your tetramer requires for stability first and then just go ahead. Couple say 1mg of antibody to NHS activated sepharose (as you probably have some sort of chromatography system, pre-packed 1ml columns (from GE) would be a good choice, and then try to bind your protein from a pre-purified extract (I'd precipitate with ammonium sulfate first). Then, follow the standard literature on affinity purification on immobilized antibodies (try to elute with low pH, alcohol gradients, NaCl, MgCl2, guanidinium salts). To make testing the eluates for your protein easier (and not only rely on UV), you might consider to label some pure protein with a dye, then you don't have to check the fractions with ELISA / westerns initially.
Is the antibody sort of purified or is it just ascites? A dilution of 1:10 is horribly low, actually. Before spending 2000 USD on 5mg antibody, you might try to have your own polyclonal, as for about 1000 USD, you'll get 2 rabbits (IgG, available almost at every 2nd corner) or, probably 2, chicken (you may end up with tons of IgY from eggs, eg from Gallus Immunotech). Protocols last about 6-8 weeks. An "orthogonal" alternative could be tagging your protein and the purifying by affinity purification to that tag or to use an immobilized ligand as bait, if applicable, but there is also no warranty that it will work perfectly. HTH Wo > I want to create an immunoaffinity column for purifying a tetrameric > recombinant protein from my cell extract (which is not precious at all; I can > grow more tissue very easily). However, the only monoclonal antibodies I can > find that react with my protein have never been tested for immunoaffinity > purification. In addition, the recommended dilutions for use in protocols > such as immunohistochemistry, western blot, and ELISA are all 1:10, which is > quite low, suggesting that the antibody's affinity for this enzyme is not > great. I will be needing around 5 mg of the pure protein per prep for my > assays. > > The antibody isn't too terribly expensive (1mg for $400). The alternative > "traditional" purification for this protein (from a different type of tissue) > is a four-column procedure (Blue Sepharose, ion exchange, hydrophobic > interaction, gel filtration). > > I am very much a novice at protein purification, so my question is, "Given > this information, do you think it is still worth attempting to make an > immunoaffinity column, or should I just try the 4 column procedure?" What > would the experts try first? > > Thanks, > > Owen _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
