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Originally published as Biophys J. BioFAST on December 21, 2004.
doi:10.1529/biophysj.104.052795
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Biophysical Journal 88:2145-2153 (2005)
© 2005 The Biophysical Society

{lambda}-Repressor Oligomerization Kinetics at High Concentrations Using Fluorescence Correlation Spectroscopy in Zero-Mode Waveguides

K. T. Samiee *, M. Foquet *, L. Guo {dagger}, E. C. Cox {dagger} and H. G. Craighead *

* School of Applied & Engineering Physics, Cornell University, Ithaca, New York 14853; and {dagger} Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544

Correspondence: Address reprint requests to Harold Craighead, C. W. Lake Jr. Professor of Engineering, Applied Physics, 205 Clark Hall, Cornell University, Ithaca, NY 14853. Tel.: 607-255-8707; E-mail: hgc1{at}cornell.edu.

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 {lambda} 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.




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