[29] Affinity chromatography: General methods

Abstract

Affinity chromatography is one of the most powerful procedures that can be applied to protein purification. The solvent system chosen for the entire affinity chromatography separation is also a critical factor to a good separation. The solvent should not degrade the sample. After choosing the affinity gel type, the resin (gel) is prepared for use. The manufacturer usually supplies the instructions needed to prepare the gel correctly. If the gel is supplied in a preswollen state, reconstituting the gel is unnecessary to obtain the full swollen volume. A wash is all that is usually required. The swollen gel is typically supplied in glycerin or similar material, which is used to help in the gel preparation and to stabilize the ligand or activated coupling complex. If the gel needs to be swollen to regain full working volume, then a swelling buffer is used prior to washing. This buffer is often a low concentration phosphate buffer (0.1 M) at or near neutral pH. Swelling times vary between 15 min and 1 hr. After swelling, the gel is washed in the buffer solution used for swelling, distilled water, or starting buffer.