532 Iv The Immune System in Health and disease To ple Buffer 30 Plastic 29 Buffer Bottom Relative mo FIGURE 23-4 Gel electrophoresis(a)A standard PAGE apparatus with cathode at the top and anode at the bottom. Samples are loaded on the top of the gel in sample wells and electrophoresis is accom- Stable plished by running a current from the cathode to the anode.(b)The obility, or distance traveled by a species during SDS. Molecules PAGE, is inversely proportional to the log of its molecular weight. The migrate to 7.0 molecular weight of a protein is readily determined by the log of its migration distance with a standard curve that plots the migration dis which their 6.0 tances of the set of standard proteins against the logs of their molecu lar weights.( c)Isoelectric focusing, or IEF, separates proteins solely by is zer 5.0 charge. Proteins are placed on a stable pH gradient and subjected to electrophoresis. Each protein migrates to its isoelectric point, the point at which its net charge is zero. Part (b) after K. Weber and M. Osbom, 975, The Proteins, 3rd ed, vol. 1, p. 179. Academic Press, gel electrophoresis(PAGE), may be used for analysis of pro- arate the components of a mixture of proteins according to teins or nudeic acids( Figure 23-4a) molecular weight. Second, because the electrophoretic mobil- In one common application, the electrophoresis of pro- ity, or distance traveled by a species during SDS-PAGE, is in- teins through a polyacrylamide gel is carried out in the pres- versely proportional to the logarithm of its molecular weight, ence of the detergent sodium dodecyl sulfate(SDS). This that distance is a measure of its molecular weight The gel is method, known as SDS-PAGE, provides a relatively simple stained with a dye that reacts with protein to visualize the and highly effective means of separating mixtures of proteins locations of the proteins. The migration distance of a protein on the basis of size. SDS is a negatively charged detergent that in question is then compared with a plot of the distances binds to protein in amounts proportional to the length of the migrated by a set of standard proteins( Figure 23-4b) protein. This binding destroys the characteristic tertiary and Another electrophoretic technique, isoelectric focusing secondary structure of the protein, transforming it into a (EF), separates proteins solely on the basis of their char negatively charged rod. A protein binds so many negatively This method is based on the fact that a molecule will move in harged SDS molecules that its own intrinsic charge becomes an electric field as long as it has a net positive or negative insignificant by comparison with the net charge of the SDs charge; molecules that bear equal numbers of positive and molecules. Therefore, treatment of a mixture of proteins with negative charges and therefore have a net charge of zero will SDS transforms them into a collection of rods whose electric not move. At most pH values, proteins(which characteristi charges are proportional to their molecular weights. This has cally bear a number of both positive and negative charges) two extremely useful consequences. First, it is possible to sep- have either a net negative or a net positive charge. However,gel electrophoresis (PAGE), may be used for analysis of pro￾teins or nucleic acids (Figure 23-4a). In one common application, the electrophoresis of pro￾teins through a polyacrylamide gel is carried out in the pres￾ence of the detergent sodium dodecyl sulfate (SDS). This method, known as SDS-PAGE, provides a relatively simple and highly effective means of separating mixtures of proteins on the basis of size. SDS is a negatively charged detergent that binds to protein in amounts proportional to the length of the protein. This binding destroys the characteristic tertiary and secondary structure of the protein, transforming it into a negatively charged rod. A protein binds so many negatively charged SDS molecules that its own intrinsic charge becomes insignificant by comparison with the net charge of the SDS molecules. Therefore, treatment of a mixture of proteins with SDS transforms them into a collection of rods whose electric charges are proportional to their molecular weights. This has two extremely useful consequences. First, it is possible to sep￾arate the components of a mixture of proteins according to molecular weight. Second, because the electrophoretic mobil￾ity, or distance traveled by a species during SDS-PAGE, is in￾versely proportional to the logarithm of its molecular weight, that distance is a measure of its molecular weight. The gel is stained with a dye that reacts with protein to visualize the locations of the proteins. The migration distance of a protein in question is then compared with a plot of the distances migrated by a set of standard proteins (Figure 23-4b). Another electrophoretic technique, isoelectric focusing (IEF), separates proteins solely on the basis of their charge. This method is based on the fact that a molecule will move in an electric field as long as it has a net positive or negative charge; molecules that bear equal numbers of positive and negative charges and therefore have a net charge of zero will not move. At most pH values, proteins (which characteristi￾cally bear a number of both positive and negative charges) have either a net negative or a net positive charge. However, 532 PART IV The Immune System in Health and Disease Apparent mass (kd) 70 10 20 30 40 50 60 0.2 0.4 0.6 0.8 1.0 Relative mobility Anode Cathode Sample wells Sample Buffer Gel Plastic frame − + + Top Mass (kd) Stable pH gradient Bottom 200 100 68 43 36 29 17 12 (a) (c) (b) Buffer Molecules migrate to position at which their net charge is zero − − − −− − + − + +− − − + pH 7.0 6.0 5.0 − 7.0 6.0 5.0 − + ++ − − − − + + + − Direction of electrophoresis FIGURE 23-4 Gel electrophoresis. (a) A standard PAGE apparatus with cathode at the top and anode at the bottom. Samples are loaded on the top of the gel in sample wells and electrophoresis is accom￾plished by running a current from the cathode to the anode. (b) The electrophoretic mobility, or distance traveled by a species during SDS￾PAGE, is inversely proportional to the log of its molecular weight. The molecular weight of a protein is readily determined by the log of its migration distance with a standard curve that plots the migration dis￾tances of the set of standard proteins against the logs of their molecu￾lar weights. (c) Isoelectric focusing, or IEF, separates proteins solely by charge. Proteins are placed on a stable pH gradient and subjected to electrophoresis. Each protein migrates to its isoelectric point, the point at which its net charge is zero. [Part (b) after K. Weber and M. Osborn, 1975, The Proteins, 3rd ed., vol. 1, p. 179. Academic Press.]