Pulsed-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 assessed for the separation of protein molecules. The allelic isoforms of apolipoprotein (a) (apo[a]) served as a model for this study because apo(a) is an unusually large protein, consisting of a variable number of repeating units. PFGE and, for comparison, conventional electrophoresis of human sera were carried out under reducing conditions in agarose gel. After blotting proteins onto nitrocellulose membrane, a combination 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 repeating units ("kringles") by both PFGE and conventional electrophoresis, but the type of agarose gel used greatly affected the speed of electrophoretic 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 single 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 protein molecules.
Csako
G
G
Clinical Pathology Department, W. G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892.
Nagy
B
B
Costello
R
R
Castelli
J C
JC
Hruskewycz
A M
AM
D001054:Apolipoproteins A / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D016521:Electrophoresis, Gel, Pulsed-Field; D006801:Humans; D018082:Kringles; D008970:Molecular Weight; D011110:Polymorphism, Genetic; D012091:Repetitive Sequences, Nucleic Acid
D001054:Apolipoproteins A