Rapid preparative isolation of major erythrocyte membrane proteins using polyacrylamide gel electrophoresis in sodium dodecylsulfate.
1. We describe a simple method for preparative, sodium dodecylsulfate/polyacrylamide gel electrophoresis of the major proteins in human erythrocyte membranes. 2. The method is based on extraction of prestained proteins from gel slabs. Three different fluorescent dyes (o-phthalaldehyde, fluorescamine and 1-dimethyl-aminonaphthalene-5-sulfonylchloride) have been used for pretaining. The method allows separation of up to 75 mg membrane protein and isolation of mg quantities of all major erythrocyte ghost proteins, while preserving the high resolution of analytical polyacrylamide gel electrophoresis. 3. Yield depends on extraction conditions and the molecular weight of the proteins being eluted. It ranges from 43-48% for protein 1 (apparent mol. wt approx. 310000) and 72-78% for protein 3(apparent mol. wt 87 000-93 000) to 87-93% for protein 6 (apparent mol. wt 35 000). 4. The labile behaviour of the high molecular "spectrin" bands (bands 1 and 2) is described. Extraction at room temperature tends to split these proteins into products of lower molecular weight. In contrast, the minor protein components 2.1 and 2.2 tend to aggregate yielding components 1 and 2. 5. N-terminal amino acid analyses have been performed on proteins 1, 2, 3, 4A, 4B, 5 and 6. Each of these bands contains several N-terminals, most of which appear constant. Some additional N-terminal amino acids vary from one donor to the next.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001798:Blood Proteins / Q000302:isolation & purification*; D002462:Cell Membrane / Q000032:analysis; D003619:Dansyl Compounds; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000032:analysis*; D005456:Fluorescent Dyes; D006801:Humans; D008722:Methods; D008970:Molecular Weight; D012967:Sodium Dodecyl Sulfate; D013997:Time Factors
D000596:Amino Acids; D001798:Blood Proteins; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D012967:Sodium Dodecyl Sulfate