POISED-FIELD GEL-ELECTROPHORESIS FOR THE SEPARATION OF LARGE PROTEIN MOLECULES EXEMPLIFIED BY THE ISOFORMS OF APOLIPOPROTEIN (A)

The performance of pulsed-field gel electrophoresis (PFGE) was assesse d for the separation of protein molecules. The allelic isoforms of apo lipoprotein (a) (apo[a]) served as a model for this study because apo( a) is an unusually large protein, consisting of a variable number of r epeating units. PFGE and, for comparison, conventional electrophoresis of human sera were carried out under reducing conditions in agarose g el. After blotting proteins onto nitrocellulose membrane, a combinatio n of monospecific rabbit anti-apo(a) antibody and alkaline phosphatase -conjugated protein A detected apo(a) isoforms in all sera tested. The various apo(a) isoforms were effectively resolved within two repeatin g units (''kringles'') by both PFGE and conventional electrophoresis, but the type of agarose gel used greatly affected the speed of electro phoretic separation. In a series of 89 human sera, 59 double-band and 30 single-band patterns were seen using either electrophoretic system. However, one specimen produced double bands with PFGE, while only a s ingle band could be detected by conventional electrophoresis, and with another specimen the opposite occurred. A total of 22 different apo(a ) isoforms were identified among these patterns. It is concluded that the increasingly available PFGE technology is a practical alternative to conventional agarose electrophoresis for the separation of large pr otein molecules.