Pore size, polyacrylamide gels, effect

N,]S2-diaHyltartardiamide (DATD) [58477-85-3] (37). The cross-linking of polymerized monomer with the comonomer is what controls the pore size of the gel polymer mesh. This level of pore size control makes polyacrylamide gel electrophoresis an effective analytical tool. 

Polyacrylamide shows many advantages over starch gel as a medium for high resolution electrophoresis and because of its synthetic nature its pore size can be more easily controlled. The gel is formed by the polymerization of the two monomers, acrylamide and a cross-linking agent, N, iV-methylene-bis-acrylamide (Figure 3.26). The proportion of the two monomers and not their total concentration is the major factor in determining the pore size, the latter having more effect on the elasticity and... 

The pore size required for a particular electrophoretic separation will depend on the difference in size of the compounds that you wish to resolve. For instance, if you wish to resolve two small proteins of 8,000 Da and 6,000 Da, you will require a small-pore-size gel for this application (—15-20% acrylamide). This same percent acrylamide gel would not permit the resolution of two larger proteins of say 150,000 Da and 130,000 Da. A larger-pore-size gel would be required for this application (—7.5-10% acrylamide). A list of components required to produce polyacrylamide gels of varying percent acrylamide is shown in Table 4-1. A list of the effective separation range of proteins on acrylamide gels made with various percent acrylamide is shown in Table 4-3. 

The pore size of a polyacrylamide gel controls the mobility and resolution of components because of the sieving effect of the pores on macromolecular species.6 The pore size may be controlled by varying the total concentrations of monomer and cross-linker, and by varying their ratio. Gel compositions are dehned by two parameters, their %T and %C values, that represent the total and crosslinker contents, respectively. These parameters are defined by Eqs. 9.5 and 9.6. 

For the latter purpose, preformed spherical beads, Biogel-P, are available from Bio-Rad Laboratories, Richmond, California. These beads are hydrophilic and chemically stable and have a uniform pore diameter and mesh size. These desired traits, and the fact that the polyacrylamide beads are readily derivatized, suggest that it should be an excellent affinity-chromatographic support. In fact, it is not often used because the heads are not sufficiently porous to permit proteins to have access to ligands hoimd within the beads. The lack of normal porosity for polyacrylamide heads is aggravated by the shrinkage of the gel occurring upon derivatization. As a result, the effective capacity of polyacrylamide gel is very low for all except the smallest enzymes. 

Effect of crosslinker concentration on gel pore structure in a 6 %T crosslinked polyacrylamide hydrogel. Evolution of (a) crosslink strand molecular weight, and (b) average pore size (L/2) estimated from Equation 5 are plotted for crosshnker concentrations ranging from 1-8 %C. Polymerization conditions T=20 °C, UV intensity = 5 W/ml. 

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