M EXPERIMENTAL FIGURE 3-32 SDS-polyacrylamide gel electrophoresis separates proteins solely on the basis of their masses. Initial treatment with SDS, a negatively charged detergent, dissociates multimeric proteins and denatures all the polypeptide chains (step 1). During electrophoresis, the SDS-protein complexes migrate through the polyacrylamide gel (step 2). Small proteins are able to move through the pores more easily, and faster, than larger proteins. Thus the proteins separate into bands according to their sizes as they migrate through the gel. The separated protein bands are visualized by staining with a dye (step 3).

unlikely to have identical net charges because their sequences, and thus the number of acidic and basic residues, are different.

In two-dimensional electrophoresis, proteins are separated sequentially, first by their charges and then by their masses (Figure 3-33a). In the first step, a cell extract is fully denatured by high concentrations (8 M) of urea and then layered on a gel strip that contains an continuous pH gradient. The gradient is formed by ampholytes, a mixture of polyanionic and polycationic molecules, that are cast into the gel, with the most acidic ampholyte at one end and the most basic ampholyte at the opposite end. A charged protein will migrate through the gradient until it reaches its isoelectric point (pI), the pH at which the net charge of the protein is zero. This technique, called iso-

electric focusing (IEF), can resolve proteins that differ by only one charge unit. Proteins that have been separated on an IEF gel can then be separated in a second dimension on the basis of their molecular weights. To accomplish this separation, the IEF gel strip is placed lengthwise on a poly-acrylamide slab gel, this time saturated with SDS. When an electric field is imposed, the proteins will migrate from the IEF gel into the SDS slab gel and then separate according to their masses.

The sequential resolution of proteins by charge and mass can achieve excellent separation of cellular proteins (Figure 3-33b). For example, two-dimensional gels have been very useful in comparing the proteomes in undifferentiated and differentiated cells or in normal and cancer cells because as many as 1000 proteins can be resolved simultaneously.

▲ EXPERIMENTAL FIGURE 3-33 Two-dimensional gel electrophoresis can separate proteins of similar mass. (a) In this technique, proteins are first separated on the basis of their charges by isoelectric focusing (step 1). The resulting gel strip is applied to an SDS-polyacrylamide gel and the proteins are separated into bands by mass (step 3). (b) In this two-

dimensional gel of a protein extract from cultured cells, each spot represents a single polypeptide. Polypeptides can be detected by dyes, as here, or by other techniques such as autoradiography. Each polypeptide is characterized by its isoelectric point (pi) and molecular weight. [Part (b) courtesy of J. Celis.]

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