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Affinity purification of antibodies

Affinity purification of antibodies

Running an affinity column.

To read about how to make a column from bacterial protein or peptides, click here.

Important information on elution conditions!!!

Different antibodies are eluted off affinity columns under different conditions. Most will come off in the glycine elution protocol outlined here. You can test if they came off or not by applying the "eluted" antibody to a small piece of nitrocellulose that has a spot of your protein on it and developing it by normal protocols. Glycine is the best option, as not only does it work with most antibodies it also causes less damage to the column itself. If it doesn't come off with glycine, try the ethanolamine base protocol, which is also not too rough of the column. If nothing comes off with either of these, try the high magnesium elution, while this will probably work it may (=probably) also destroy your column.

Preparation of serum