An isoelectric focusing procedure for erythrocyte membrane proteins and its use for two-dimensional electrophoresis |
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| Authors: | P A Dockham R C Steinfeld C J Stryker S W Jones G A Vidaver |
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| Affiliation: | 1. Department of Biochemistry, 1 King''s College Circle, University of Toronto, Toronto M5S 1A8, Canada;2. Department of Biochemistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton, Alberta T6G 2H7, Canada;3. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK;4. Division of Molecular Biosciences, Imperial College London, London, SW7 2AZ, UK |
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| Abstract: | Procedures are described and evaluated for one-dimensional isoelectric focusing of erythrocyte membrane dissolved in lysine, urea, and Triton X-100 without using sodium dodecyl sulfate (SDS) and for two-dimensional electrophoresis with SDS in the second dimension. The membrane was completely dissolved, most of the proteins including the anion porter(s) entered the focusing gel, and complex, well-resolved patterns were seen. Ampholines, 2-mercaptoethanol, or SDS in the applied sample each seriously reduced focusing resolution and phenylmethylsulfonyl fluoride blurred the patterns. The two-dimensional patterns showed more and sharper spots than did patterns obtained from membrane initially dissolved with SDS. Anion porter spots were seen with both procedures. However, major cytoskeletal proteins were much less well recovered with the former procedure than with the latter. |
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