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Abstract
A discontinuous electrophoretic system for the isolation of membrane proteins from
acrylamide gels has been developed using equipment for sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE). Coomassie dyes were introduced to induce a charge
shift on the proteins and aminocaproic acid served to improve solubilization of membrane
proteins. Solubilized mitochondria or extracts of heart muscle tissue, lymphoblasts,
yeast, and bacteria were applied to the gels. From cells containing mitochondria,
all the multiprotein complexes of the oxidative phosphorylation system were separated
within one gel. The complexes were resolved into the individual polypeptides by second-dimension
Tricine-SDS-PAGE or extracted without SDS for functional studies. The recovery of
all respiratory chain complexes was almost quantitative. The percentage recovery of
functional activity depended on the respective protein complex studied and was zero
for some complexes, but almost quantitative for others. The system is especially useful
for small scale purposes, e.g., separation of radioactively labeled membrane proteins,
N-terminal protein sequencing, preparation of proteins for immunization, and diagnostic
studies of inborn neuromuscular diseases.