Sedimentation equilibrium-quantitative polyacrylamide gel electrophoresis (SE-QPAGE): a new technique for the...

A new technique for the characterization of self- and hetero-associations in multicomponent solutions without chemical modification of the solutes is described. Solutions containing between one and four distinct protein components were first centrifuged to sedimentation equilibrium in swinging bucket rotors. Following centrifugation, the contents of each centrifuge tube were fractionated into 3-microliter aliquots deriving from sequential laminae of solution, and each aliquot was diluted into sodium dodecyl sulfate-containing buffer. Following incubation, 20 microliters of each aliquot was applied to individual lanes of a polyacrylamide gel. Following electrophoresis, proteins on the gel were stained with Coomassie blue R-250. The pattern of stained bands on the gel was digitized and the resulting image file analyzed to yield relative intensities of individual bands. A quantitative relationship between band intensity and amount of protein in a band was established by means of calibration gels. This relationship was found to be essentially constant for a variety of different proteins except for a protein-dependent scaling factor. Analysis of the dependence of band intensity of a single protein component upon the radial position of the original lamina from which the fraction was derived yielded reliable estimates of the molecular weight of that component in the original solution and in some cases showed that two components were cosedimenting as a complex.