Isoelectric focusing of sparingly soluble proteins in immobilized pH gradients, exemplified by microvillar membrane hydrolases

A novel fractionation technique is described for analysis of membrane-bound enzymes and sparingly soluble proteins: isoelectric focusing in a mixed-type matrix, containing a primary, immobilized pH gradient with a superimposed, secondary carrier ampholyte pH gradient. Three microvilli hydrolases: dipeptidyl peptidase IV, gamma-glutamyl transferase and alkaline phosphatase exhibit an array of sharply focused, enzyme active bands in the pH 4-6.5 range. The separation pattern obtained is by far superior to any separation achieved by either technique separately.