Free mobility determination by electrophoresis in polyacrylamide containing agarose at a nonrestrictive concentration.
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of Ferguson plots from the lowest gel concentrations that give a mechanically stable gel to 0% T has recently been replaced by measurement of mobilities across that concentration range, using the addition of 0.5% agarose to polyacrylamide at the various low concentrations in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).
Pospichal
J
J
Section on Macromolecular Analysis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
Vicchio
D
D
Chrambach
A
A
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004247:DNA / Q000737:chemistry*; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006454:Hemoglobins / Q000737:chemistry; D008970:Molecular Weight; D010799:Phycoerythrin / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012685:Sepharose
D006454:Hemoglobins; D011506:Proteins; D010799:Phycoerythrin; D004247:DNA; D012685:Sepharose