Analysis for Purity

Abstract

Resolution of separate components of a protein mixture is achieved most clearly using an electrophoretic method. As discussed in Section 8.2, preparative electrophoresis has problems in the design and use of apparatus, so despite its obvious potential, it is not often used. But in an analytical mode, electrophoresis is a most widely used method; indeed, it is almost obligatory to characterize a purified protein preparation by an electrophoretic technique. Analytical electrophoresis in a gel system requires only 5–25 µg protein (or less with sensitive silver-staining techniques); this is rarely a significant proportion of what is available. Before gel systems were developed, electrophoretic analysis was carried out in the Tiselius free-boundary apparatus, requiring tens of milligrams of protein. This did not resolve closely similar proteins, and analy a single sample required a great deal of effort and attention. Paper and other cellulose-based supports were introduced for zone analytical electrophoresis, which eliminated two of the disadvantages of the Tiselius apparatus; only small amounts of protein were needed, and the technique was easy, requiring only simple equipment. But resolution was scarcely any better even on the superior cellulose acetate strips, because separation is based only on a rough charge/size ratio; many proteins move together as a single peak (see Section 8.2).