What happens when a mixture of SDS-solubilized proteins is run through a slab of polyacrylamide gel? Each protein molecule binds large numbers of the negatively charged detergent molecules, which mask the protein’s intrinsic charge and cause it to migrate toward the positive electrode when a voltage is applied. Proteins of the same size tend to move through the gel with similar speeds because (1) their native structure is completely unfolded by the SDS, so that their shapes are the same, and (2) they bind the same amount of SDS and therefore have the same amount of negative charge. Larger proteins, with more charge, are subjected to larger electrical forces but also to a larger drag. In free solution, the two effects would cancel out, but, in the mesh of the polyacrylamide gel, which acts as a molecular sieve, large proteins are retarded much more than small ones. As a result, a complex mixture of proteins is fractionated into a series of discrete protein bands arranged in order of molecular weight (Figure 8–13). The major proteins are readily detected by staining the proteins in the gel with a dye such as Coomassie blue. Even minor proteins are seen in gels treated with a silver stain, so that as little as 10 ng of protein can be detected in a band. For some purposes, specific proteins can also be labeled with a radioactive isotope tag; exposure of the gel to film results in an autoradiograph on which the labeled proteins are visible (see Figure 8–16).
SDS-PAGE is widely used because it can separate all types of proteins, including those that are normally insoluble in water—such as the many proteins in membranes. And because the method separates polypeptides by size, it provides information about the molecular weight and the subunit composition of proteins. Figure 8–14 presents a photograph of a gel that has been used to analyze each of the successive stages in the purification of a protein.
Because different proteins can have similar sizes, shapes, masses, and overall charges, most separation techniques such as SDS polyacrylamide-gel electrophoresis or ion-exchange chromatography cannot typically separate all the proteins
Figure 8–12 The detergent sodium dodecyl sulfate (SDS) and the reducing agent β-mercaptoethanol. These two chemicals are used to solubilize proteins for SDS polyacrylamide-gel electrophoresis. The SDS is shown here in its ionized form.
sample loaded onto gel protein with two by pipette subunits, A and B, joined by a disulfde single-subunit bridge protein
Figure 8–13 SDS polyacrylamide-gel electrophoresis (SDS-PAGe). (A) An electrophoresis apparatus. (B) Individual polypeptide chains form a complex with negatively charged molecules of sodium dodecyl sulfate (SDS) and therefore migrate as a negatively charged SDS–protein complex through a porous gel of polyacrylamide. Because the speed of migration under these conditions is greater the smaller the polypeptide, this technique can be used to determine the approximate molecular weight of a polypeptide chain as A well as the subunit composition of a protein. If the protein contains a large amount of carbohydrate, however, it will move anomalously on the gel and its apparent molecular weight estimated by SDS-PAGE will be misleading. Other modifications, such as phosphorylation, can also cause small changes in a protein’s migration in the gel. slab of polyacrylamide gel in a cell or even in an organelle. In contrast, two-dimensional gel electrophoresis, which combines two different separation procedures, can resolve up to 2000 proteins in the form of a two-dimensional protein map.