Optimization of large gel 2D electrophoresis for proteomic studies of skeletal muscle
Wellstone Center for FSHD
Adult; Animals; Cells, Cultured; Electrophoresis, Gel, Two-Dimensional; Humans; Molecular Weight; Muscle Fibers, Skeletal; Muscle Proteins; Muscle, Skeletal; Myoblasts; Proteolysis; Proteomics; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Sensitivity and Specificity; Solubility; Thiourea; Urea
Cell Biology | Developmental Biology | Molecular Biology | Molecular Genetics | Musculoskeletal Diseases | Nervous System Diseases
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.
DOI of Published Version
Reed, P. W., Densmore, A. and Bloch, R. J. (2012), Optimization of large gel 2D electrophoresis for proteomic studies of skeletal muscle. Electrophoresis. 2012 Apr;33(8):1263-70. doi: 10.1002/elps.201100642. Link to article on publisher's site
Reed, Patrick W.; Densmore, Allison; and Bloch, Robert J., "Optimization of large gel 2D electrophoresis for proteomic studies of skeletal muscle" (2012). Wellstone Center for FSHD Publications. 12.