Preparative isoelectric focusing in immobilized pH gradients. I. General principles and methodology

A new method for preparative protein purification is described, based on the use of Immobiline matrices. After electrofocusing, the protein zone of interest is recovered by electrophoretic transfer to a hydroxyapatite gel, from which it is eluted with 0.2 M phosphate buffer, pH 6.8, with yields for the proteins studied in the range 76-98%. For six different proteins, the focusing step gives a common upper limit of approximately 45 mg protein/ml gel as mean concentration in a focused protein zone. It is demonstrated that in practical preparative work, components with a pI difference of 0.007 pH units can be completely resolved, and that on a 5-mm-thick gel of dimensions 240 X 110 mm, samples containing as much as 400 mg of the major protein component can be applied. Focusing of large amounts of a salt-containing sample is demonstrated with the aid of human serum. A theoretical expression is given relating the concentration distribution and maximum protein concentration within a focused zone to the applied voltage, the pH slope used and the zone width. Based on this expression and the finding of an upper concentration limit for a protein we shown how to optimize the parameters in preparative work with immobilized pH gradients in relation to the separation power needed. Finally, it is shown that, in comparison with conventional preparative electrofocusing in polyacrylamide gels, immobilized pH gradients allow a ten-fold increase in load, whilst still giving a resolution comparable to that of analytical isoelectric focusing.