The resolution between two native proteins and between their sodium dodecyl sulfate-complexes in agarose and polyacrylamide gel electrophoresis.

Abstract

Commercial gel electrophoresis apparatus with intermittent fluorescence scanning of the migration path (HPGE-1000 apparatus, LabIntelligence) makes it possible to measure band width and migration distance as a function of the duration of electrophoresis. As a result, resolution can be evaluated quantitatively and therefore different gel media can be compared objectively. The resolution of fluorescein carboxylate labeled conalbumin (molecular mass 86 kDa) and soybean trypsin inhibitor (22.7 kDa) in gel electrophoresis was found to increase as a function of the gel type in the order SeaKem GTG-, SeaKem Gold-agarose, 2% N,N'-methylenebisacrylamide cross-linked polyacrylamide, MetaPhor-XR-, and SeaPrep-agarose. The advantage in resolving capacity of SeaPrep agarose over the polyacrylamide gel was by a factor of up to five. The resolving capacity of the agaroses was in indirect relation to the degree of electroendosmosis. In all media, resolution increased with migration distance (time). The same proteins when reacted with sodium dodecyl sulfate (SDS) resolve (i) better at up to 6% SeaPrep agarose concentration than in polyacrylamide, as in the gel electrophoresis of the native proteins; (ii) less effectively, by contrast, at SeaPrep agarose concentrations > 6%, than in polyacrylamide gel; and (iii) significantly better in 4-6% SeaPrep agarose than in 4-6% SeaKem GTG agarose. Since Ferguson plot analysis in both agarose and polyacrylamide gels shows that the two SDS-proteins are larger than the native proteins with which they are complexed, the superiority of polyacrylamide gels above 7% appears to be correlated with the fact that its mean pore radius, estimated for both media using identical assumptions and identical rigid spherical standards - proteins, is approximately seven times larger than that of SeaPrep agarose in the concentration range of 3-8%, and that therefore the molecular "fit" in polyacrylamide is closer than that in SeaPrep agarose of the concentration range used. The dependence of resolution on the ratio of particle radius to mean pore radius ("fit") is also suggested by the fact that the two SDS-proteins resolve in a biphasic dependence on gel concentration in both agarose and polyacrylamide, with a maximum at 6% agarose and 10% polyacrylamide.