Isoelectric focusing in layers of granulated gels II. Preparative isoelectric focusing

Abstract

A method for preparative isoelectric focusing of 0.1–10 g amounts of proteins is described. For anticonvective stabilization of the pH gradient, layers of granulated gels (e.g. Sephadex or Bio-Gel) of variable length, width and thickness were used either on glass plates or in troughs. Load capacity, defined as the amount of protein per ml gel suspension, was determined to be 5–10 mg per ml for total protein, irrespective of the pH range of the carrier ampholytes. For single proteins load capacities of 0.25–1 mg per ml were found for pH 3–10 carrier ampholytes, and 2–4 mg per ml for narrow pH range ampholytes. Experiments on a quartz plate followed by densitometric evaluation in situ at 280 nm have demonstrated that it is possible to proceed from analytical thin-layer isoelectric focusing to preparative separations without loss of resolution, just by changing the dimension of the gel layer and increasing the protein load. Improved resolution which facilitates isolation of isoelectrically homogeneous components could be achieved on a 40 cm long separation distance. The geometry of a layer is favourable to heat dissipation and thus permits the use of high voltage gradients. Recovery of the focused proteins is high and elution simple. The efficiency of the method is illustrated by examples showing separations of single proteins and protein mixtures.