Electrophoresis Separates Molecules on the Basis of Their Charge Mass Ratio

Electrophoresis is a technique for separating molecules in a mixture under the influence of an applied electric field. Dissolved molecules in an electric field move, or migrate, at a speed determined by their charge:mass ratio. For example, if two molecules have the same mass and shape, the one with the greater net charge will move faster toward an electrode.

SDS-Polyacrylamide Gel Electrophoresis Because many proteins or nucleic acids that differ in size and shape have nearly identical charge:mass ratios, electrophoresis of these macromolecules in solution results in little or no separation of molecules of different lengths. However, successful separation of proteins and nucleic acids can be accomplished by electrophoresis in various gels (semisolid suspensions in water) rather than in a liquid solution. Electrophoretic separation of proteins is most commonly performed in polyacrylamide gels. When a mixture of proteins is applied to a gel and an electric current is applied, smaller proteins migrate faster through the gel than do larger proteins.

Gels are cast between a pair of glass plates by polymerizing a solution of acrylamide monomers into polyacry-lamide chains and simultaneously cross-linking the chains into a semisolid matrix. The pore size of a gel can be varied by adjusting the concentrations of polyacrylamide and the cross-linking reagent. The rate at which a protein moves through a gel is influenced by the gel's pore size and the strength of the electric field. By suitable adjustment of these parameters, proteins of widely varying sizes can be separated.

In the most powerful technique for resolving protein mixtures, proteins are exposed to the ionic detergent SDS (sodium dodecylsulfate) before and during gel electrophoresis (Figure 3-32). SDS denatures proteins, causing multimeric proteins to dissociate into their subunits, and all polypeptide chains are forced into extended conformations with similar charge:mass ratios. SDS treatment thus

▲ EXPERIMENTAL FIGURE 3-31 Centrifugation techniques separate particles that differ in mass or density. (a) In differential centrifugation, a cell homogenate or other mixture is spun long enough to sediment the denser particles (e.g., cell organelles, cells), which collect as a pellet at the bottom of the tube (step 2|). The less dense particles (e.g., soluble proteins, nucleic acids) remain in the liquid supernatant, which can be transferred to another tube (step 3). (b) In rate-zonal centrifugation, a mixture is spun just long enough to separate molecules that differ in mass but may be similar in shape and density (e.g., globular proteins, RNA molecules) into discrete zones within a density gradient commonly formed by a concentrated sucrose solution (step 2). Fractions are removed from the bottom of the tube and assayed (step 5).

eliminates the effect of differences in shape, and so chain length, which corresponds to mass, is the sole determinant of the migration rate of proteins in SDS-polyacrylamide electrophoresis. Even chains that differ in molecular weight by less than 10 percent can be separated by this technique. Moreover, the molecular weight of a protein can be estimated by comparing the distance that it migrates through a gel with the distances that proteins of known molecular weight migrate.

Two-Dimensional Gel Electrophoresis Electrophoresis of all cellular proteins through an SDS gel can separate proteins having relatively large differences in mass but cannot resolve proteins having similar masses (e.g., a 41-kDa protein from a 42-kDa protein). To separate proteins of similar masses, another physical characteristic must be exploited. Most commonly, this characteristic is electric charge, which is determined by the number of acidic and basic residues in a protein. Two unrelated proteins having similar masses are

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