FIGURE 3-19 Electrophoresis. (a) Different samples are loaded in wells or depressions at the top of the polyacrylamide gel. The proteins move into the gel when an electric field is applied. The gel minimizes convection currents caused by small temperature gradients, as well as protein movements other than those induced by the electric field. (b) Proteins can be visualized after electrophoresis by treating the gel with a stain such as Coomassie blue, which binds to the proteins but not to the gel itself. Each band on the gel represents a different pro-

tein (or protein subunit); smaller proteins move through the gel more rapidly than larger proteins and therefore are found nearer the bottom of the gel. This gel illustrates the purification of the enzyme RNA polymerase from E. coli. The first lane shows the proteins present in the crude cellular extract. Successive lanes (left to right) show the proteins present after each purification step. The purified protein contains four subunits, as seen in the last lane on the right.

Isoelectric focusing is a procedure used to determine the isoelectric point (pI) of a protein (Fig. 3-21). A pH gradient is established by allowing a mixture of low molecular weight organic acids and bases (ampholytes; p. 81) to distribute themselves in an electric field generated across the gel. When a protein mix ture is applied, each protein migrates until it reaches the pH that matches its pI (Table 3-6). Proteins with different isoelectric points are thus distributed differently throughout the gel.

Combining isoelectric focusing and SDS electrophoresis sequentially in a process called two-dimensional

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