Two-dimensional electrophoresis of basic proteins with equilibrium isoelectric focusing in carrier ampholyte-pH gradients

✍ Scribed by Dr. Thierry Rabilloud

• Publisher: John Wiley and Sons
• Year: 1994
• Tongue: English
• Weight: 600 KB
• Volume: 15
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/elps.1150150147

✦ Synopsis

Paris equilibrium iSOeleCtI'k focusing in Cader ampholyte-

A modified procedure for the two-dimensional electrophoretic analysis of basic polypeptides is described. This method uses isoelectric focusing with carrier ampholytes in the first dimension, and sodium dodecyl sulfate-electrophoresis in the second dimension. Counteraction of the cathodic drift is achieved by glass tube treatment (silanization), electrolyte modification (use of weak bases and acids), protection of the catholyte from carbon dioxide, and the addition of glycerol to the gel mix. Better resolution and reproducibility are obtained than with nonequilibrium pH gradient electrophoresis, since quasi equilibrium focusing can be obtained. DDT, dithiothreitol; IEF, isoelectric focusing; IPG, immobilized pH gradient; PAGE, polyacrylamide gel electrophoresis; PDA, piperazine lytes ( l O h Pharmalyte 6.5-9 4-' Oh Phamalyte 8-10'5). diacrylamide; SDS, sodium dodecyl sulfate; 2-D, two-dimensional; The gel mix was degassed for 5 min (water pump with %T, grams of monomers (including cross-linker) per 100 mL of gel; stirring of the mixture) and polymerization was initiated TEMED, N,N,K,K-tetramethylethylenediamine