Instability of pH gradients in isoelectric focusing on polyacrylamide gel

Abstract

During isoelectric focusing in polyacrylamide gel of a crude human pancreatic extract, the pH gradient and the position of protein bands were found to be unstable with time in accordance with previous work (2,6). Instability of pH gradients in p I 3–10 or 3–6 Ampholine was monitored by measurement of pH changes and protein band displacement at the “isoelectric end point.” In p I 3–10 gels the results are consistent with the hypothesis that pH gradient instability is caused by a progressive electrophoretic migration of Ampholine from the neutral gel center toward the gel periphery. However, in a p I 3–6 gradient an acidic protein band was found to be displaced toward the cathode, indicating that displacement cannot occur by electrophoresis only. Instability is increased as a function of voltage (2) and decreased as a function of viscosity (6) and Ampholine concentration. It is not due to the presence of protein in the gel, to migration of Ampholine out of the gel, or to protein or Ampholine modification under the conditions of vinyl polymerization (8). It occurs regardless of whether the acid or basic (8) reservoir is positioned on top of the gel.