Estimation of polyacrylamide gel pore size from Ferguson plots of linear DNA fragments. II. Comparison of gels with different crosslinker concentrations, added agarose and added linear polyacrylamide

Abstract

The mobilities of various DNA fragments in two normally migrating molecular weight ladders were studied in polyacrylamide gels containing different concentrations of the crosslinker N,N′‐methylenebisacrylamide (Bis). The acrylamide concentration ranged from 2.5–10.5%T (w/v); the Bis concentration ranged from 0.5–10%C (w/w), with respect to total acrylamide. Ferguson plots were constructed for each of the DNA fragments in gels of each composition. The Ferguson plots of the different multimers in each molecular weight ladder were nearly parallel in gels containing 0.5–3%C, converged close to a common intercept at zero gel concentration in gels containing 4%C, and crossed at ∼ 1.5%T in gels containing 5 and 10%C. If the mobilities observed for the different DNA fragments at zero gel concentration were also extrapolated to zero DNA molecular weight, a common limiting mobility was observed in gels of all crosslinker concentrations. This limiting mobility was approximately equal to the free solution mobility of DNA. The effective pore radius of each gel was estimated from Ferguson plots based on relative mobilities, using the mobility of the smallest DNA fragment in each molecular weight ladder as the reference mobility. The calculated gel pore radii ranged from 142 nm to 19 nm, respectively, for gels containing 4.6%T, 1.5%C, and 10.5%T, 5 or 10%C. These pore radii are an order of magnitude larger than previously accepted values, but are consistent with scanning electron microscope measurements (Rüchel, R., et al., J. Chromatogr. 1978, 42, 77–90). In gels containing 3–10%C, the calculated pore radii decreased with increasing gel concentration approximately as T^−0.5, suggesting that these gels may be long fiber or “1‐D” gels. The pore radii of gels with lower crosslinker concentrations decreased more slowly with increasing polyacrylamide concentration. Pore sizes were also determined for gels containing 3% Bis and 0.5% w/v agarose, and 3% Bis plus 2% w/w high molecular weight linear polyacrylamide. The pore radii of the mixed agarose‐polyacrylamide gels were only about half as large as those observed in gels of the same composition without agarose. The inclusion of 2% w/w linear polyacrylamide in the polymerization mixture decreased the effective pore size of the polyacrylamide matrix by about 10%.