In the first step, the proteins are separated by their intrinsic charges. The sample is dissolved in a small volume of a solution containing a nonionic (uncharged) detergent, together with β-mercaptoethanol and the denaturing reagent urea. This solution solubilizes, denatures, and dissociates all the polypeptide chains but leaves their intrinsic charge unchanged. The polypeptide chains are then separated in a pH gradient by a procedure called isoelectric focusing, which takes advantage of the variation in the net charge on a protein molecule with the pH of its surrounding solution. Every protein has a characteristic isoelectric point, the pH at which the protein has no net charge and therefore does not migrate in an electric field. In isoelectric focusing, proteins are separated electrophoretically in a narrow tube of polyacrylamide gel in which a gradient of pH is established by a mixture of special buffers. Each protein moves to a position in the gradient that
Figure 8–14 Analysis of protein samples by SDS polyacrylamide-gel electrophoresis. The photograph shows a Coomassie-stained gel that has been used to detect the proteins present at successive stages in the purification of an enzyme. The leftmost lane (lane 1) contains the complex mixture of proteins in the starting cell extract, and each succeeding lane analyzes the proteins obtained after a chromatographic fractionation of the protein sample analyzed in the previous lane (see Figure 8–10). The same total amount of protein (10 μg) was loaded onto the gel at the top of each lane. Individual proteins normally appear as sharp, dye-stained bands; a band broadens, however, when it contains a large amount of protein. (From
T. Formosa and B.M. Alberts, J. Biol. Chem. 261:6107–6118, 1986.) mass(daltons)100,00040,00015,000
Figure 8–15 Separation of protein molecules by isoelectric focusing.
at low pH,
At low pH (high H+ concentration), the charged the protein carboxylic acid groups of proteins tend to at the isoelectric example, –NH3+), giving most proteins point, the protein a net positive charge. At high pH, the and therefore no carboxylic acid groups are negatively at high pH, charged (–COO–) and the basic groups the electric feld; tend to be uncharged (for example, –NH2), for the protein giving most proteins a net negative charge.
shown, thethe protein
At its isoelectric pH, a protein has no net is 6.5 charge since the positive and negative charges balance. Thus, when a tube containing a fixed pH gradient is subjected to a strong electric field in the appropriate direction, each protein species migrates until it forms a sharp band at its isoelectric corresponds to its isoelectric point and remains there (Figure 8–15). This is the pH, as shown.
first dimension of two-dimensional polyacrylamide-gel electrophoresis.
In the second step, the narrow tube gel containing the separated proteins is again subjected to electrophoresis but in a direction that is at a right angle to the direction used in the first step. This time SDS is added, and the proteins separate according to their size, as in one-dimensional SDS-PAGE: the original tube gel is soaked in SDS and then placed along the top edge of an SDS polyacrylamide-gel slab, through which each polypeptide chain migrates to form a discrete spot. This is the second dimension of two-dimensional polyacrylamide-gel electrophoresis. The only proteins left unresolved are those that have both identical sizes and identical isoelectric points, a relatively rare situation. Even trace amounts of each polypeptide chain can be detected on the gel by various staining procedures—or by autoradiography if the protein sample was initially labeled with a radioisotope (Figure 8–16). The technique has such great resolving power that it can distinguish between two proteins that differ in only a single charged amino acid, or a single negatively charged phosphorylation site.