Free mobility determination by electrophoresis in polyacrylamide containing agarose at a nonrestrictive concentration

Abstract

In the determination of the free mobility, related to the surface net charge, by quantitative gel electrophoresis, the previous arbitrary extrapolation of Ferguson plots from the lowest gel concentrations that give a mechanically stable gel to 0% T has recently been replaced by measurement of mobilities across that concentration range, using the addition of 0.5% agarose to polyacrylamide at the various low concentrations in application to a DNA fragment 155 bp in size (Orbán, L. et al., in preparation). The present study applies that approach to several proteins and DNA fragments smaller than 1300 bp, using 0.4% agarose in polyacrylamide gels of varying concentration. The intercepts of the plots with the mobility axis provide experimental data by which the free mobility in polyacrylamide gel electrophoresis can be estimated for molecules not significantly retarded in their migration at the agarose concentration admixed to polyacrylamide. Across the gel concentration range below 3% T, in the presence of agarose, the Ferguson plots of proteins and DNA fragments are convex. It was shown by mass spectrometry that this convex curvature of the plots in the mixed polymer is not significantly due to low polymerization efficiency in the concentration range of liquid polyacrylamide (below 3%T).

Detection of conformational and net charge differences in DNA‐protein complexes by quantitative electrophoresis on polyacrylamitie‐agarose copolymer gels

“…2), by contrast to the linearity of these plots for the free DNA fragments. The convex curvature in plots derived from polyacrylamide matrices containing a constant, small concentration of agarose is not protein specific, as was originally thought, but was found in another study [36] to be common to proteins and DNAin excess of 600 bp * Calculations based on data reported by [34].…”

Commercial automated gel electrophoresis apparatus: Application to DNA, band dispersion, nonlinear Ferguson curves, and isolation

“…If at such gel concentrations oxygen inhibits gelation in contact with air, as in the present procedure of stacking gel formation [19] or in an enlarged sample applicator ("concentrator") should be difficult to achieve, polymerization in an atmosphere of argon, under submersion of silicone oil [20,211 or in presence of 0.4% agarose [22] is indicated.…”

Simultaneous Ferguson plot analysis, using electrophoresis on a single agarose pore gradient gel, of DNA fragments contained in a mixture

“…Five microliters of sample solution (2.08 1. 18 DNA/gel tube) were applied. Field strength was measured during electrophoresis as described [ 161.…”