Methods for sample labeling and meniscus determination in the fluorescence-detected analytical ultracentrifuge.
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.
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.
Michael F
MF
Bailey
Lauren M
LM
Angley
Matthew A
MA
Perugini
D001896:Boron Compounds / Q000032:analysis*; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D004256:DNA Polymerase I / Q000032:analysis* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000032:analysis*; D011485:Protein Binding; D014461:Ultracentrifugation / Q000379:methods*
C527198:4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D004247:DNA; D004256:DNA Polymerase I