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Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins. Anal Biochem 1989 Aug 01;180(2):205-10

Date

08/01/1989

Pubmed ID

2817350

DOI

10.1016/0003-2697(89)90116-4

Abstract

Electrophoresis of the high-molecular-mass proteins (greater than 500 kDa) of muscle myofibrils is difficult using conventional procedures. The mobility of these proteins was influenced by the heating time in sample buffer, the use of 2-mercaptoethanol in the upper reservoir buffer, and the pH of the resolving gel in a stacking sodium dodecyl sulfate gel system. Heating samples for 4 min (versus shorter times), addition of 2-mercaptoethanol to the upper reservoir buffer, and reducing the pH of the resolving gel to 8.6 all enhanced the mobility and resolution of the high-molecular-weight proteins on polyacrylamide gels. The sulfhydryl reducing agents commonly used in protein sample buffers (2-mercaptoethanol and dithiothreitol) were found to migrate at the electrophoretic dye front. Inclusion of 10 mM 2-mercaptoethanol in the upper reservoir buffer or blocking free sulfhydryl groups with N-ethylmaleimide prevented intermolecular disulfide bond formation during electrophoresis. The addition of 10 mM 2-mercaptoethanol to the buffer used for electroblotting also improved efficiency of protein transfer to nitrocellulose.

Author List

Fritz JD, Swartz DR, Greaser ML

Author

Jeffery Duane Fritz PhD Associate Professor in the Medical School Regional Campuses department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Animals
Buffers
Cattle
Collodion
Disulfides
Dithiothreitol
Electrophoresis, Polyacrylamide Gel
Gels
Hot Temperature
Hydrogen-Ion Concentration
Mercaptoethanol
Molecular Weight
Muscle Proteins
Myofibrils
Oxidation-Reduction
Protein Denaturation
Rabbits
Reproducibility of Results
Time Factors