Technical and Developmental Details of Basic Technique

Any ion will undergo electrophoresis to migrate in an electric field. Proteins are complex polyions with a net charge at all pH values other than its isoelectric point. Problems associated with convective disturbance in free solution led early researchers to consider various supporting media for electrophoresis such as paper, cellulose acetate and various thin layer materials where the separation depends largely on the charge density at a given pH. The reader should refer to the separate articles on Electrophoresis Theory and Cellulose Acetate Electrophoresis for further details. Other early workers considered the properties of various gels where the pore size approximates the size of the protein molecules themselves leading to a separation based on both charge and molecular size. The extent of molecular sieving depends on the pore size of the gel being used. For example, the pore size of agarose gels is sufficiently large that sieving of most proteins is minimal, whereas larger DNA molecules are sieved very well. Again, for a discussion of this refer to the separate article on Agarose Elec-trophoresis.

The pore size of polyacrylamide gels may be changed in a systematic and reproducible fashion by varying the percentage of monomer and crosslinker to give a matrix which maximizes the molecular sieving effect for a wide range of proteins and the reader should refer to the separate article on Polyacrylamide Gel Electrophoresis (PAGE) for a discussion on varying porosity in this medium.

Native proteins, however, often occur as multiple supramolecular assemblies of many peptide sub-units in different configurations affected by non-covalent bonding, particularly in the case of membrane proteins. Shapiro et al. (1967) first demonstrated the potential of the superior protein dissociating qualities of sodium dodecyl sulfate (SDS) in an electrophoretic system designed to separate individual polypeptides on the basis of their molecular weight alone but the definitive publication in this area is undoubtedly that of Laemmli (1970) who first combined a discontinuous buffer system (see separate article on Discontinuous Electrophoresis) with the use of SDS in sample preparation and gel electrophor-esis. Protein complexes are solubilized and dissociated with such high efficiency in 2% SDS and 5% mercaptoethanol that typically over 90% of the protein in a crude lysate will enter the gel matrix and be resolved.

In the discontinuous system, proteins are dissolved by denaturing treatment at 100°C with the dissociating agents SDS and mercaptoethanol in a Tris-HCl buffer at pH 6.8. Gels are constructed in two stages both containing 0.1% SDS. The separating gel in the original Laemmli publication was formed using 30% stock acrylamide monomer with 0.8% bisacrylamide as a cross-linker. A final solution was made to 8 or 10% acrylamide containing 0.375 m Tris-HCl pH 8.8. The resolving (separating) gel is polymerized using tetramethylenediamine (TEMED) (catalyst) and ammonium persulfate (free radical initiator). A 'stacking' gel (at 3% acrylamide) is then cast on top of the resolving gel in the same manner but containing 0.125 m Tris-HCl at the same pH as the buffer in which the protein mixtures were dissociated (pH 6.8). The electrode buffer contains 0.025 m Tris/0.192M glycine to a pH of 8.3 also with 0.1% SDS. Upon electrophoresis, protein anions in the form of rod-shaped SDS complexes are compressed in the stacking gel between the leading chloride ions and the trailing glycinate ions which, because of the pH difference between buffer systems, progressively close the gap as electrophoresis proceeds. (Again, see the separate articles on Discontinuous Electrophoresis and Iso-tachophoresis). The result is a concentration or stacking of the SDS-protein anions as extremely sharp bands ( + 5-10 |im) behind the leading chloride ion in strict order of mobility. These complexes then enter the separating gel and since, supposedly, the charge-mass ratio is invariant (see later for a caveat) are separated by molecular sieving according to their molecular size only. Gels of particular acrylamide concentration and therefore pore size may be calibrated using standard proteins of known molecular weight. By extrapolation, reliable molecular weight estimates of large numbers of polypeptides in a complex mixture may be obtained. Proteins are fixed in the gel after electrophoresis using a 50% trich-loroacetic acid solution and stained in a Coomassie blue solution. Radiolabelled proteins were also detected by autoradiography. For a more detailed discussion please refer to the separate article on Detection Techniques, Staining, Autoradiography and Blotting.

In the original Laemmli procedure, gels were elec-trophoresed in small glass cylinders and since the absolute mobility of SDS-polypeptides varied slightly from gel to gel the relative mobility of standard and unknown bands was calculated as the ratio between the mobility of the protein and the mobility of the tracking dye, bromphenol blue (BPB), which travels with the SDS-micelle front behind the leading buffer front.

A major improvement on the original procedure uses rectangular slab gels of uniform thickness (typi cally 0.5-1.5 mm) with sample wells (typically 10-30) set into the stacking gel. Electrophoretic apparatus was originally constructed according to a variety of ad hoc patterns, in house, from perspex, using gels polymerized between notched glass plates, although now most laboratories use commercially available equipment. The use of such equipment (see the separate article on Slab Gel Electrophoresis: Equipment) has led to highly standardized reliable separations since standards and unknowns may be run under identical conditions. The apparent molecular weights of unknowns may be obtained by extrapolation from a plot of log MWt vs. mobility of standard proteins. Figure 1 shows how individual

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Molecular weight (kDa)

Figure 1 (See Colour Plate 41). The inset shows the separation of human head hair proteins using 12% sodium dodecyl sulfate-polyacrylamidegel electrophoresis with the point of migration of standard proteins to the left under the heading kDa: lanes 1-3 S-carboxymethylated proteins; lane 4 reduced non-S-carboxmethylated proteins: lane 1 14C Autoradiograph; lanes 2 and 4, silver stain; lane 3, Coomassie stain. The main body of Figure 1 shows a densitometric profile of the separated lane 1 (autoradiograph 14C-S-carboxymethylated, green). Lane 2 (silver stain, S-carboxymethylated, red); lane 4 (silver stain, non-S-carboxymethylated). Molecular weights were extrapolated from a plot of log MWt vs. relative migration of standard proteins.

10 20 50 100 200

Molecular weight (kDa)

Figure 1 (See Colour Plate 41). The inset shows the separation of human head hair proteins using 12% sodium dodecyl sulfate-polyacrylamidegel electrophoresis with the point of migration of standard proteins to the left under the heading kDa: lanes 1-3 S-carboxymethylated proteins; lane 4 reduced non-S-carboxmethylated proteins: lane 1 14C Autoradiograph; lanes 2 and 4, silver stain; lane 3, Coomassie stain. The main body of Figure 1 shows a densitometric profile of the separated lane 1 (autoradiograph 14C-S-carboxymethylated, green). Lane 2 (silver stain, S-carboxymethylated, red); lane 4 (silver stain, non-S-carboxymethylated). Molecular weights were extrapolated from a plot of log MWt vs. relative migration of standard proteins.

zones in a complex mixture of human hair keratins may all be assigned an apparent molecular weight and how the entire molecular weight profile is apparently shifted 20-40 kDa higher after S-carboxy-methylation.

Although several alternative sample extraction and buffer systems are available (see later), the use of the basic Laemmli system has proved so robust and reliable that it has revolutionized protein characterization in complex mixtures to the extent that proteins of widely differing function are routinely described according to their apparent molecular mass on Laemmli SDS-PAGE. The explosive increase in the use of this technique across a comprehensive range of the biological sciences is illustrated in Figure 2 showing the number of citations each year of the Laemmli (1970) publication.

I will now discuss subsequent major methodological developments of the basic Laemmli protocol.

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