PHOSPHORIMAGER ENHANCEMENT OF SEDIMENTATION EQUILIBRIUM-QUANTITATIVE POLYACRYLAMIDE-GEL ELECTROPHORESIS - A HIGHLY SENSITIVE TECHNIQUE FOR QUANTITATION OF EQUILIBRIUM GRADIENTS OF INDIVIDUAL COMPONENTS IN MIXTURES

The technique called sedimentation equilibrium-quantitative polyacryla mide gel electrophoresis (Darawshe ct al. (1993) Anal. Biochem. 215, 2 36-242) has been extended to permit the quantitation and analysis of g radients of individual radiolabeled components in a mixture of radiola beled solutes centrifuged to sedimentation equilibrium. Immediately fo llowing centrifugation, the contents of a sample tube are fractionated into aliquots corresponding to laminae of solution at different radia l positions in the centrifuge. Following treatment with sodium dodecyl sulfate-containing buffer, a portion of each fraction is subjected to electrophoresis on a polyacrylamide gel. The gel is then incubated wi th a storage phosphor plate and subsequently scanned with a Molecular Dynamics PhosphorImager. The concentration of an individual radiolabel ed component at a particular radial distance is proportional to the in tegrated intensity of the image of the radiolabeled band of that compo nent in the fraction corresponding to that radial distance, Concentrat ion gradients reconstructed in this fashion are interpreted in the con text of conventional sedimentation equilibrium theory. The results of control experiments carried out with purified proteins of known molar mass and the measurement of the molar mass of a new, partially purifie d protein are reported. (C) 1995 Academic Press, Inc.