Isoelectric Focusing: pH Gradients Established with Simple Buffers and a Cation-Selective Membrane

Abstract

A method for isoelectric focusing is presented in which pH gradients are established with simple buffer systems in a thin granulated gel bed. The gel bed is supported by a cation-selective membrane over a buffer-filled channel with a sloping bottom. The component of the electric field parallel to the gel and membrane surfaces that results from this arrangement may be considered fixed at each position in the gel bed but increases with the decreasing depth of the buffer filled channel below. This feature, together with the ion-selective properties of the supporting membrane, allows the electric field to maintain a shallow pH gradient by adjusting the composition of the buffer at each position in the gel bed. Ribonuclease, myoglobin, β-lactoglobulin, and ovalbumin, representing proteins with a wide distribution of isoelectric points, were focused in the buffers glycine, imidazole, Mes, and acetic acid, respectively. Focused protein fractions were rerun on conventional polyacrylamide-carrier ampholyte gels.