title: Optimization of large gel 2D electrophoresis for proteomic studies of skeletal muscle.
abstract: We describe improved methods for large format, two-dimensional gel electrophoresis (2DE) that improve protein solubility and recovery, minimize proteolysis, and reduce the loss of resolution due to contaminants and manipulations of the gels, and thus enhance quantitative analysis of protein spots. Key modifications are: (i) the use of 7 M urea and 2 M thiourea, instead of 9 M urea, in sample preparation and in the tops of the gel tubes; (ii) standardized deionization of all solutions containing urea with a mixed bed ion exchange resin and removal of urea from the electrode solutions; and (iii) use of a new gel tank and cooling device that eliminate the need to run two separating gels in the SDS dimension. These changes make 2DE analysis more reproducible and sensitive, with minimal artifacts. Application of this method to the soluble fraction of muscle tissues reliably resolves ~1800 protein spots in adult human skeletal muscle and over 2800 spots in myotubes.
authors.affiliation: The Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, Department of Physiology, University of Maryland School of Medicine University of Maryland, Baltimore, MD 21201, USA. preed001@umaryland.edu
authors.forename: Patrick W
authors.initials: PW
authors.lastname: Reed
authors.forename: Allison
authors.initials: A
authors.lastname: Densmore
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Bloch
mesh_terms: D000328:Adult; D000818:Animals; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D008970:Molecular Weight; D018485:Muscle Fibers, Skeletal / Q000737:chemistry; D009124:Muscle Proteins / Q000032:analysis* / Q000737:chemistry; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000166:cytology; D032446:Myoblasts / Q000737:chemistry; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012995:Solubility; D013890:Thiourea / Q000737:chemistry; D014508:Urea / Q000737:chemistry
chemical_list: D009124:Muscle Proteins; D014508:Urea; D013890:Thiourea