The method of immobilized pH gradient of CIEF used in protein separation

An immobilized pH gradient method had been developed for proteins separation by two-step capillary isoelectric focusing based on chemical bonding. The double functional reagent, N,N′-carbonyldiimidazole was introduced in the capillary as a linkage in the immobilization reactions. Moreover, the commercial ampholytes (CAs) were distributed according to their pI values in the condition of electric field, and then connected with N,N′- carbonyldiimidazole by chemical bonding to realize the immobilized pH gradient in capillary. A test sample containing a protein mixture of hemoglobin (Hb), bovine serum albumin (BSA) and pepsin was separated by using this method.