Affinity Electrophoresis

 By analogy to affinity chromatography, it is possible to introduce specific ligands for a macromolecule into the gels of gel electrophoresis and to measure the specific retardation of the macromolecule due to its interaction with such a reagent. The advantage of such affinity methods lies in the augmented resolving power conferred by the specificity of the binding interaction.

The procedures used to introduce affinity reagents into gels have varied. In cross electrophoresis, a ligand with a net charge opposite to the species of interest migrates electrophoretically into the gel in the opposite direction. Alternatively, uncharged ligands can simply be added to the gelation mixture.

Macromolecular substrates within a gel may serve as immobilized affinity reagents, either by themselves or as carriers of covalently attached affinity groups. The magnitude of the electrophoretic retardation depends on the concentration of the affinity reagent in the gel; quantitative determination of this relationship makes it possible to estimate the apparent association constant for binding of the ligand to the sample. Further information concerning the interaction can be gained from affinity electrophoresis by variation of the buffer composition (eg, the addition of metal ions to the buffer), the pH, or the temperature.