monoclonal antibody suitable for immunoaffinity column?

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Wed Oct 12 03:28:33 EST 2011


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



More information about the Methods mailing list