Advantages and Disadvantages of Carrier Ampholyte IEF

Abstract

There are several advantages to the use of carrier ampholytes for running IEF separations. Tube gels using carrier ampholytes are easy to prepare and do not require sophisticated gradient casting equipment. Ampholyte mixtures can be simply blended and optimized for wide or more limited pH ranges. Blends can be engineered to be linear throughout the pH range or sigmoidal to emphasize a particular pH region (1). Protein resolution is exquisite when very thin tube gels are used and the reproducibility of the gradients can be excellent (2–4). Some of the disadvantages of carrier ampholyte IEF have centered on batch-to-batch reproducibility of the ampholytes themselves, since the chemical synthesis procedure is long and complicated. On the other hand, the same caveat exists for immobilized pH gradients, since each new batch of IPG acylamido/immobilines is subject to lot-to-lot variability as well. Another disadvantage of carrier ampholytes is that although theoretically the pH gradient is stable when it reaches “equilibrium,” in reality, the phenomenon of cathodic drift owing to electroendosmosis in the gel causes an eventual breakdown of the basic end of the pH gradient with time (5). However, the effect can be minimized if the IEF separations are standardized with respect to volt hours. If very basic proteins (i.e., with pIs above 8.0) are to be separated, nonequilibrium pH electrophoresis or NEpHGE, should be employed.