Slab Gel Electrophoresis

Subsequently naturally occurring gels were investigated particularly for the separation of plasma proteins. Agar gels, especially in the form of the highly purified subunit agarose, are still a popular medium for protein separations. Agar forms gels at relatively low concentrations, but due to its high number of sulfate and carboxylic groups it generates a significant EOF. The sulfate and carboxylic fraction can be removed chemically to leave agarose, which has much reduced EOF and gives a better resolution of proteins. The gels are formed as slabs by pouring a liquid gel into a mould and letting it set before placing in an electrophoresis system.

In 1955 Smithies, modifying an earlier method, introduced the technique of starch gel electrophoresis and its superior resolving power for proteins was immediately apparent. The starch was derived from hydrolysed potato starch and poured to a thickness of 5-10 mm. In addition, the pores in the starch gel matrix are close to the molecular size of proteins so that in this form electrophoretic movement is accompanied by some molecular sieving, but the effect was irreproducible.

In 1955 Raymond and Weintraub introduced a synthetic polymer gel made from the monomer polyacrylamide, as a replacement for starch. Poly-acrylamide gels have many advantages over starch being tougher, more flexible, naturally clearer and chemically inert. Acrylamide gels are formed by the polymerization of the monomer acrylamide in the presence of an N,N'-methylenebisacrylamide (BIS). The reaction requires initiators and a catalyst (cross-linking reagent). Commonly used catalysts are ammonium or potassium persulfate (for chemical polymerization) or light and riboflavin (for photo-polymerization). A common initiator is N,N,N',N'-tetramethylethyldiamine (TEMED). The polymerization should be performed at above 20°C to prevent incomplete polymerization. The reaction takes place via vinyl polymerization and gives a randomly coiled gel structure. The concentration of polyacrylamide can be varied over a wide range without making the gels unmanageable. The pore size can also be controlled exactly by varying the amount of BIS used in the polymerization (Table 3) so the precision of the molecular sieving is enhanced.

The use of such gels for electrophoresis is commonly referred to as polyacrylamide gel electrophor-esis (PAGE). In addition to the tight control of pore size other advantages of polyacrylamide gels compared to starch gels are that the adsorption of macro-molecules to polyacrylamide is negligible, there is little EOF associated with polyacrylamide and strong,

Table 3 Effect of cross-linking on pore size of acylamide gels

Percentage of

Molecular weight

N, N'-methylenebisacrylamide (BIS)

range resolved

5

50 000-300 000

10

10 000-100 000

15

& 5 000

but thin, transparent gels can be cast permitting faster separation. A disadvantage is that the monomers are toxic and need to be handled with caution.

Development of the gel system followed rapidly. In 1964 Ornstein and Davis simultaneously introduced discontinuous (DISC) electrophoresis, which improved both the solubility of the proteins in the gel as well as improving the resolution. In DISC elec-trophoresis the gel is formed in two sections, a stacking gel and a resolving gel. The resolving gel has small pores filled with a buffer of pH 8.8 high mobility buffer (e.g. 2-amino-2-hydroxy-methylpropane-1,3-diol-hydrochloric acid (TRIS-HCl)) and a large pore stacking gel contains a buffer of about pH 6.8. The sample is loaded at approximately pH 8.8. These conditions induce the proteins to migrate according to isotachophoresis through the stacking gel, then stacked at the interface with the resolving gel before slowly destacking and resolving as they pass through that gel.

The size and shape of macromolecules complicates their separation, so special buffer/electrolyte conditions are employed. Non-dissociating (native) buffer systems, as described earlier for DISC electrophoresis, are used to separate the native forms of proteins and double-stranded DNA. Dissociating (denaturing) buffer systems can also be used, for example, double-stranded DNA is denatured using urea, formamide, sodium hydroxide or intercalating agents such as ethidium bromide prior to application to the gel.

Although it is appreciated that the use of gels not only aided the electrophoretic separation, for macro-molecules it also introduced a size-sieving effect equivalent to gel filtration in chromatography. This can be used to 'size' molecules, particularly proteins. So if the conditions are correct and if the pores are of the appropriate dimensions could PAGE also be used to determine the molecular weight of proteins?

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