ANALYTICAL BIOCHEMISTRY 233, 31–35 (1996) Article No. 0003 Enhanced Electrophoretic Separation and Resolution of Myosin Heavy Chains in Mammalian and Avian Skeletal Muscles Eric R. Blough,* Erik R. Rennie,* Fanjie Zhang,† and Peter J. Reiser* , † ,1 *Department of Exercise Science and †Department of Oral Biology, The Ohio State University, Columbus, Ohio 43210 Received May 1, 1995 status, and in response to neuromuscular disease (1). Due We report a sodium dodecyl sulfate – polyacrylamide to these reasons and because of its critical physiological gel electrophoresis protocol for the reliable separa- importance, assessment of MHC isoform expression is es- tion, with high resolution, of myosin heavy chain iso- sential for a further understanding of skeletal muscle con- forms in adult avian (chicken) and mammalian tractile properties during development, aging, and disease. (mouse) skeletal muscles. The sample preparation Fast and slow MHC isoforms from avian (5) and mam- time can be relatively short, thereby minimizing en- malian (6) muscle were first successfully separated using dogenous proteolytic activity which may otherwise re- SDS–PAGE employing acrylamide percentages between sult in dispersed and spurious bands. Inclusion of 2- 5 and 7.5%. Danieli-Betto et al. (7) were able to resolve mercaptoethanol in the upper electrode buffer greatly both the IIA and IIB MHCs in rat skeletal muscle using improves band resolution. Glycerol is commonly in- 6% SDS–PAGE after increasing the gel glycerol content cluded in the reported protocols for myosin heavy to 40%. Several laboratories have separated another mam- chain separation and our results demonstrate that the malian MHC isoform, referred to as IID or IIX (8–10), concentration of glycerol employed can have a marked using gradient SDS–PAGE, or after extensive myofibril effect on the relative order of migration among myosin (11) or myosin isolation (12, 13). The gels utilized in these heavy chain isoforms.  1996 Academic Press, Inc. studies consisted of low percentages of acrylamide and high glycerol concentrations, both of which compromise the mechanical properties of the gel. Alteration of glycerol concentration and pH and the inclusion of 2-mercaptoetha- Skeletal muscles are composed of a large number of nol have been shown to alter the electrophoretic mobility fibers with different physiological and biochemical proper- and resolution of large myofibrillar proteins ( ú500 kDa) ties (1). The myosin heavy chain (MHC) 2 molecule is the (14, 15). To our knowledge, observations concerning MHC most abundant myofibrillar protein on a weight basis and separation and these variables have not been adequately plays a crucial role in muscle contraction. For example, described. A simple method of electrophoretic separation maximum velocity of shortening of single muscle fibers of MHC isoforms is reported here. Adjustment of the acryl- (V max ) is strongly correlated with MHC isoform composi- amide percentage and glycerol concentration in the gels, tion (2). Skeletal muscle demonstrates a remarkable abil- along with the inclusion of 2-mercaptoethanol in the elec- ity to alter its protein expression during development and trode buffer, allows for consistent separation of four mouse in response to a variety of stimuli. The MHC molecule is and seven chicken MHCs. encoded by a multigene family (3) and MHC expression has been shown to proceed in a tissue-specific and develop- METHODS mentally regulated manner (4). Alterations in MHC ex- pression have been found subsequent to changes in con- Preparation of Muscle Samples tractile activity, circulating thyroid level, innervation All animals from which muscle samples were ob- tained for this study were cared for and sacrificed in 1 To whom correspondence should be addressed. accordance with an institutionally approved animal 2 Abbreviations used: MHC, myosin heavy chain; DTT, dithiothrei- care and use protocol. A muscle is quickly removed and tol; bis, N,N-methylene-bis(acrylamide); TEMED, N,N,N,N-tetra- methyethylenediamine. placed in cold relaxing solution which has the following 31 0003-2697/96 $12.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.