Isoelectric Focusing and Immunodetection of Plasma Antithrombin

The first studies of plasma protein variation relied predominantly on conventional electrophoretic methods in media such as starch, agar, or cellulose acetate, which separated molecules on the basis of their relative differences in net charge and size. The introduction of isoelectric focusing in polyacrylamide gels (IEF/PA) led to the discovery of additional variability previously undetected by conventional electrophoretic techniques. IEF/PA separates molecules on the basis of their isoelectric points (pI) in a pH gradient generated by the application of an electric field to a mixture of buffer components, known as carrier ampholytes, which are present within a polyacrylamide matrix. Thus, it is possible to discriminate between molecules having a difference in pI as little as 0.01 pH units. The exact composition of isolectric focusing gels may be varied depending on the protein under investigation. In particular, alternative pH gradients may be generated by mixing ampholytes in the appropriate pH range. When used in combination with a simple “native” blotting procedure (1 ), the basic IEF/PA protocol described below has proven successful in our hands for studying microheterogeneity of plasma antithrombin and, with minor modifications to optimize results, it may be used to analyze any acidic protein of interest, provided a specific antiserum is available.