Affinity Chromatography Proteins

Affinity Chromatography of Proteins

Affinity Chromatography:  Affinity Techniques that Allows Purification of Receptor Proteins

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Purifying cell-surface hormone receptors often can be identified and followed through isolation procedures by affinity labeling.  In this technique, cells are mixed with an excess of radio labeled hormone to saturate the hormone-binding sites on its specific receptor.  After unbound hormone is washed away, the mixture is treated with a chemical agent that covalently cross-links the bound labeled hormone to the receptor.  Most cross-linking agents contain two groups that react with free amino groups; by reacting with an amino group in the receptor with one in the bound ligand, the cross-linking agent covalently joins the receptor and ligand.  A radio labeled ligand that is cross-linked to its receptor remains bound even in the presence of detergents and other denaturing agents that are used to solubilize receptor proteins from the cell membrane. 

An important technique used in purifying cell-surface receptors that retain their hormone-binding ability when solubilized is affinity chromatography.  In this technique, a ligand of the receptor of interest is chemically linked to polystyrene beads.  A crude, detergent-solubilized preparation of membrane proteins is passed through a column containing these beads.  Only the receptor binds to the beads; the other proteins are washed through the column by excess fluid.  When an excess of the ligand is passed through the column, the bound receptor is displaced from the beads and eluted from the column.  This technique is similar to the principle to antibody affinity chromatography, except that hormone ligand rather than an antibody is attached to column beads.  In some cases, a hormone receptor can be purified as much as 100,000-fold in a single affinity chromatographic step.

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