Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins.
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.
Fritz
J D
JD
Muscle Biology Laboratory, University of Wisconsin, Madison 53706.
Swartz
D R
DR
Greaser
M L
ML
D000818:Animals; D002021:Buffers; D002417:Cattle; D003101:Collodion; D004220:Disulfides / Q000032:analysis; D004229:Dithiothreitol / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005782:Gels; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008623:Mercaptoethanol / Q000032:analysis; D008970:Molecular Weight; D009124:Muscle Proteins / Q000032:analysis*; D009210:Myofibrils / Q000032:analysis*; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D011817:Rabbits; D015203:Reproducibility of Results; D013997:Time Factors
D002021:Buffers; D004220:Disulfides; D005782:Gels; D009124:Muscle Proteins; D008623:Mercaptoethanol; D003101:Collodion; D004229:Dithiothreitol