Pulse electrophoresis of muscle myosin heavy chains in sodium dodecyl sulfate-polyacrylamide gels.
We have developed a new method that provides enhanced resolution of myosin heavy chain (MHC) isoforms by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The key feature of this protocol involves the application of current to slab SDS gels in a pulsatile, repetitive manner rather than continuously as in standard gel systems. This protocol, designated pulse electrophoresis, was achieved by means of a device that intermittently gates the output of a conventional power supply. When used in long (32 cm) separating gels, pulse electrophoresis not only significantly improves the resolution of MHC isoforms compared to conventional systems, but also reduces common artifacts associated with long running times, such as blurred bands and comingling of closely spaced bands. In addition to the increased resolution of protein bands, pulse electrophoresis also allows detection of bands corresponding to previously unidentified MHC isoforms in mammalian and avian tissue. In rat myocardium, for example, pulse electrophoresis revealed three MHC isoform bands, two of which appeared to correspond to two alpha-MHC subspecies. Alternative splicing of the rat alpha-MHC gene is known to generate two isoform species differing by inclusion (or exclusion) of a single glutamine residue, whose relative levels of expression correspond nicely with the amounts of each band identified in this study. Therefore, we cannot rule out that the system presented here may be sufficiently sensitive to differentiate between high molecular weight proteins differing in a single amino acid.
Sant'Ana Pereira
J A
JA
Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
Greaser
M
M
Moss
R L
RL
D000818:Animals; D016477:Artifacts; D002645:Chickens; D004581:Electronics; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D005895:Gizzard, Avian / Q000737:chemistry; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000737:chemistry*; D009206:Myocardium / Q000737:chemistry*; D018995:Myosin Heavy Chains / Q000032:analysis* / Q000737:chemistry; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry; D051381:Rats; D013552:Swine
D020033:Protein Isoforms; D018995:Myosin Heavy Chains