This protocol describes antibody purification using a peptide affinity column. Peptides can be designed that use naturally occurring cysteines within the protein target's primary sequence, or a cysteine can be added to either end of the peptide to provide free thiols for attachment. The peptides can then be covalently attached to resins bearing thiol-reactive linkers. The most commonly used thiol-reactive moieties are iodoacetyl and maleimide, both of which react selectively with peptides containing cysteine thiols. Although gravity can be used to cycle the antibody solution (e.g., serum) over the column (it is recommended that the antibody be cycled multiple times to obtain maximal yield), the use of a pump to apply the serum to the column in a continuous flow manner improves the yield of antibody. Similarly, washing the column after application of the antibody without and with 0.5 m NaCl should be performed with at least 20 column volumes.
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