monoclonal antibody suitable for immunoaffinity column?

Owen Pierce via methods%40net.bio.net (by omp from sfu.ca)
Tue Oct 11 15:26:47 EST 2011


Dear Experts,


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



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