PHOSPHORIMAGER ENHANCEMENT OF SEDIMENTATION EQUILIBRIUM-QUANTITATIVE POLYACRYLAMIDE-GEL ELECTROPHORESIS - A HIGHLY SENSITIVE TECHNIQUE FOR QUANTITATION OF EQUILIBRIUM GRADIENTS OF INDIVIDUAL COMPONENTS IN MIXTURES
S. Darawshe et al., PHOSPHORIMAGER ENHANCEMENT OF SEDIMENTATION EQUILIBRIUM-QUANTITATIVE POLYACRYLAMIDE-GEL ELECTROPHORESIS - A HIGHLY SENSITIVE TECHNIQUE FOR QUANTITATION OF EQUILIBRIUM GRADIENTS OF INDIVIDUAL COMPONENTS IN MIXTURES, Analytical biochemistry, 229(1), 1995, pp. 8-14
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.