Phosphorlmager Enhancement of Sedimentation Equilibrium-Quantitative Polyacrylamide Gel Electrophoresis: A Highly Sensitive Technique for Quantitation of Equilibrium Gradients of Individual Components in Mixtures

✍ Scribed by S. Darawshe; N. Merezhinskaya; A.P. Minton

• Publisher: Elsevier Science
• Year: 1995
• Tongue: English
• Weight: 654 KB
• Volume: 229
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1995.1371

✦ Synopsis

The technique called sedimentation equilibrium-quantitative polyacrylamide gel electrophoresis (Darawshe et al. (1993) Anal. Biochem. 215, 236-242) has been extended to permit the quantitation and analysis of gradients of individual radiolabeled components in a mixture of radiolabeled solutes centrifuged to sedimentation equilibrium. Immediately following centrifugation, the contents of a sample tube are fractionated into aliquots corresponding to laminae of solution at different radial 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 with a strong phosphor plate and subsequently scanned with a Molecular Dynamics PhosphorImage. The concentration of an individual radiolabeled component at a particular radial distance is proportional to the integrated intensity of the image of the radiolabeled band of that component in the fraction corresponding to that radial distance. Concentration gradients reconstructed in this fashion are interpreted in the context 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 purified protein are reported.