λ-repressor oligomerization kinetics at high concentrations using fluorescence correlation spectroscopy in zero-mode waveguides

K. T. Samiee, M. Foquet, L. Guo, E. C. Cox, Harold G. Craighead

Research output: Contribution to journalArticle

82 Scopus citations

Abstract

Fluorescence correlation spectroscopy (FCS) has demonstrated its utility for measuring transport properties and kinetics at low fluorophore concentrations. In this article, we demonstrate that simple optical nanostructures, known as zero-mode waveguides, can be used to significantly reduce the FCS observation volume. This, in turn, allows FCS to be applied to solutions with significantly higher fluorophore concentrations. We derive an empirical FCS model accounting for one-dimensional diffusion in a finite tube with a simple exponential observation profile. This technique is used to measure the oligomerization of the bacteriophage λ repressor protein at micromolar concentrations. The results agree with previous studies utilizing conventional techniques. Additionally, we demonstrate that the zero-mode waveguides can be used to assay biological activity by measuring changes in diffusion constant as a result of ligand binding.

Original languageEnglish (US)
Pages (from-to)2145-2153
Number of pages9
JournalBiophysical Journal
Volume88
Issue number3
DOIs
StatePublished - Mar 2005

ASJC Scopus subject areas

  • Biophysics

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