Sodium Dodecyl Sulfate SDS Electrophoresis

SDS electrophoresis fractionates polypeptide chains essentially on the basis of their size. It is therefore a simple, yet powerful and reliable, method for molecular mass determination. In 1967 Shapiro et al. first reported that electrophoretic migration in SDS is proportional to the effective molecular radius and, thus, to the Mr of the polypeptide chain. This means that SDS must bind to proteins and cancel out differences in molecular charge, so that all components will migrate solely according to size. Surprisingly, large amounts of SDS appear to be bound (an average of 1.4 g SDS/g protein). This means that the number of SDS molecules bound is of the order of half the number of amino acid residues in a polypeptide chain. This amount of highly charged surfactant molecules is sufficient to overwhelm effectively the intrinsic charges of the polymer coil, so that their net charge per unit mass becomes approximately constant. If migration in SDS (and disulfide-reducing agents, such as 2-mercaptoethanol, in the denaturing step, for a proper unfolding of the proteins) is proportional only to Mr, then, in addition to cancelling out charge differences, SDS also equalizes molecular shape differences (e.g. globular vs rod-shaped molecules). This seems to be the case for protein-SDS mixed micelles. These complexes can be assumed to behave as ellipsoids of constant minor axis (c. 1.8 nm) and a major axis proportional to the length of the amino acid chain (i.e. to molecular mass) of the protein. The rod length for the 1.4 g SDS/g protein

Figure 4 Ferguson plots (log Rm, relative mobility, vs %T, total monomer concentration) in the case of: (A) lactic dehydrogenase (LDH) 1 and 2 (isomers of charge, exhibiting the same mass); (B) serum albumin (polymeric forms, from monomer to heptamer, having constant charge and pure size difference, since all curves meet in gel-free environment, at 2% T where polyacrylamide will liquefy); (C) ferritin and ovalbumin, two totally unrelated proteins differing in both size and charge. (Parts (A) and (C) reproduced with permission from Hedrickand Smith, 1968 and Part (B) from Thorun, 1971.)

Figure 4 Ferguson plots (log Rm, relative mobility, vs %T, total monomer concentration) in the case of: (A) lactic dehydrogenase (LDH) 1 and 2 (isomers of charge, exhibiting the same mass); (B) serum albumin (polymeric forms, from monomer to heptamer, having constant charge and pure size difference, since all curves meet in gel-free environment, at 2% T where polyacrylamide will liquefy); (C) ferritin and ovalbumin, two totally unrelated proteins differing in both size and charge. (Parts (A) and (C) reproduced with permission from Hedrickand Smith, 1968 and Part (B) from Thorun, 1971.)

complex is of the order of 0.074 nm per amino acid residue. For further information on detergent properties, see Helenius and Simons (1975).

In SDS electrophoresis, the proteins can be prelabelled with dyes that covalently bind to their -NH2 residues. The dyes can be conventional, like the blue dye Remazol, or fluorescent, such as dansyl chloride, fluorescamine, O-phthaldialdehyde, and MDPF (2-methoxy-2,4-diphenyl-3[2H]-furanone). Prelabelling is compatible with SDS electrophoresis, as the size increase is minimal, but would be anathema in disc electrophoresis or IEF, as it would generate a series of bands of slightly altered mobility or pI from an otherwise homogeneous protein. Although at its inception SDS electrophoresis used continuous buffers, today the preferred set up is via discontinuous buffers and matrices, simplified from the original disc electrophoresis assembly (see Figure 5). This ensures much higher resolving power, due to formation of ultrathin protein zones.

For treatment of data, the sample and Mr standards are electrophoresed side-by-side in a gel slab. After detection of the polypeptide zones, the migration distance (or RF) is plotted against log Mr to produce a calibration curve (Neff et al., 1981) from which the Mr of the sample can be calculated (see Figure 6). It should be noted that in a gel of constant %T, linearity is obtained only in a certain range of molecular sizes. Outside this limit a new gel matrix of appropriate porosity should be used. Two classes of proteins show anomalous behaviour in SDS electrophoresis: glyco-proteins (because their hydrophilic oligosaccharide units prevent hydrophobic binding of SDS micelles) and strongly basic proteins, e.g. histones (because of electrostatic binding of SDS micelles through their sulfate groups). The first anomaly can be partially alleviated by using alkaline Tris/borate buffers, which will increase the net negative charge on the glycoprotein and thus produce migration rates well correlated with molecular size. The migration of hi-stones can be improved by using pore-gradient gels and allowing the polypeptide chains to approach the pore limit.

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Solar Panel Basics

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