Methods for sample labeling and meniscus determination in the fluorescence-detected analytical ultracentrifuge

Anal Biochem. 2009 Jul 15;390(2):218-20. doi: 10.1016/j.ab.2009.03.045. Epub 2009 Apr 5.

Abstract

The fluorescence detection system for the analytical ultracentrifuge (AU-FDS) enables the measurement of hydrodynamic properties and interactions of biomolecules at subnanomolar concentrations. In this study, we describe methods for (i) preparing and purifying fluorescently labeled biomolecules and (ii) determining the meniscus position in the AU-FDS using BODIPY 493/503 fluorescent dye suspended in light oil. We subsequently use these methods to measure the interaction of DNA with Escherichia coli Klenow fragment (KF) and show that KF binds matched DNA to form 1:1 and 2:1 (protein/DNA) complexes with dissociation constants of 4.2 and 22 nM, respectively.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Boron Compounds / analysis*
  • DNA / analysis*
  • DNA / metabolism
  • DNA Polymerase I / analysis*
  • DNA Polymerase I / metabolism
  • Escherichia coli / enzymology*
  • Fluorescent Dyes / analysis*
  • Protein Binding
  • Ultracentrifugation / methods*

Substances

  • 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene
  • Boron Compounds
  • Fluorescent Dyes
  • DNA
  • DNA Polymerase I