Gel Electrophoresis Separates Proteins by Size and Charge

When subjected to an electric field in an electrophoresis chamber, a charged molecule will move toward the oppositely charged electrode. The rate at which a charged molecule moves in a stable field (its electrophoretic mobility) depends upon two factors specific to the molecule: one is the sign and magnitude of its net electrical charge, and the other is its size and shape. All other factors being equal, if molecules are of equal size the one with higher net charge will move faster in an applied electrical field due to the molecular seiving properties of the solid medium. It also follows that small molecules will move faster than large ones of the same net charge. Although there are exceptions in which the shape of a molecule may increase or decrease its frictional drag and cause atypical migration behavior, these general principles underlie all electrophoretic separations.

Most electrophoretic separations are not conducted in free solution but rather in a stable supporting medium, such as a gel. The most popular in reseach laboratories is a polymerized and crosslinked form of acrylamide. Separation on polyacrylamide gels, commonly referred to as poly acrylamide

Biotin active ester

Biotinylated Ab

Labeled avidin ulJ

Avidin bound to biotinylated Ab

FIGURE 23-3

Labeling of antibody with biotin. An antibody preparation is mixed with a biotin ester, which reacts with the antibody. The biotin-labeled antibody can be used to detect antigens on a solid substrate such as the well of a microtiter plate. After washing away unbound antibody, the bound antibody can be detected with labeled avidin. The avidin can be radioactively labeled or linked to an enzyme that catalyzes a color reaction, as in ELISA procedures (see Figure 6-10).

Cathode Q-

Plastic frame

Sample wells

Sample wells

Buffer o -C

200 100 68

43 36 29

17 12

60 50

60 50

0.2 0.4 0.6 0.8 Relative mobility

Bottom

0.2 0.4 0.6 0.8 Relative mobility

Stable PH gradient

Stable PH gradient

Molecules pH migrate to 7.0

position at ->

which their 6.0 net charge is zero

Molecules pH migrate to 7.0

position at ->

which their 6.0 net charge is zero

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 accomplished 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 distances of the set of standard proteins against the logs of their molecular 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. Osbom, 1975, The Proteins, 3rd ed, vol. 1, p. 179. Academic Press.]

gel electrophoresis (PAGE), may be used for analysis of proteins or nucleic acids (Figure 23-4a).

In one common application, the electrophoresis of proteins through a polyacrylamide gel is carried out in the presence 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 mobility, or distance traveled by a species during SDS-PAGE, is inversely 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 characteristically bear a number of both positive and negative charges) have either a net negative or a net positive charge. However, for each protein there is a particular pH, called its isoelectric point (pi), at which that protein has equal numbers of positive and negative charges. Isoelectric focusing makes use of a gel containing substances, called carrier ampholytes, that arrange themselves into a continuous pH gradient when subjected to an electric field. When a mixture of proteins is applied to such a gel and subjected to electrophoresis, each protein moves until it reaches that point in the gradient where the pH of the gel is equal to its isoelectric point. It then stops moving because it has a net charge of zero. Isoelectric focusing is an extremely gentle and effective way of separating different proteins (Figure 23-4c).

A method known as two-dimensional gel electrophoresis (2D gel electrophoresis) combines the advantages of SDS-PAGE and isoelectric focusing in one of the most sensitive and discriminating ways of analyzing a mixture of proteins. In this method, one first subjects the mixture to isoelectric focusing on an IEF tube gel, which separates the molecules on the basis of their isoelectric points without regard to molecular weight. This is the first dimension. In the next step, one places the IEF gel lengthwise across the top of an SDS-polyacrylamide slab (that is, in place of the sample wells in Figure 23-4a) and runs SDS-PAGE. Preparatory to this step, all proteins have been reacted with SDS and therefore migrate out of the IEF gel and through the SDS-PAGE slab according to their molecular weights. This is the second dimension. The position of the proteins in the resulting 2D gel can be visualized in a number of ways. In the least sensitive the gel is stained with a protein-binding dye (such as Coomassie blue). If the proteins have been radiolabeled, the more sensitive method of autoradiography can be used. Alternatively, silver staining is a method of great sensitivity that takes advantage of the capacity of proteins to reduce silver ions to an easily visualized deposit of metallic silver. Finally, immuno-blotting—blotting of proteins onto a membrane and detection with antibody (see Figure 6-13)—can be used as a way of locating the position of specific proteins on 2D gels if an appropriate antibody is available. Figure 23-5 shows an autoradiograph of a two-dimensional gel of labeled proteins from murine thymocytes.

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  • jole
    What is electrophoretic mobility of molecule moving in sds page?
    8 years ago

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