TY - JOUR
T1 - λ-repressor oligomerization kinetics at high concentrations using fluorescence correlation spectroscopy in zero-mode waveguides
AU - Samiee, K. T.
AU - Foquet, M.
AU - Guo, L.
AU - Cox, E. C.
AU - Craighead, Harold G.
N1 - Funding Information:
This work was supported by the Department of Eergy, by the Defense Advanced Research Projects Agency, and by the National Institutes of Health (NIH HG001506). Fabrication was done at the Cornell Nano-Scale Science and Technology Facility supported by National Science Foundation grant ECS-9731293, its users, Cornell University, and industrial partners.
PY - 2005/3
Y1 - 2005/3
N2 - 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.
AB - 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.
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U2 - 10.1529/biophysj.104.052795
DO - 10.1529/biophysj.104.052795
M3 - Article
C2 - 15613638
AN - SCOPUS:21244458506
VL - 88
SP - 2145
EP - 2153
JO - Biophysical Journal
JF - Biophysical Journal
SN - 0006-3495
IS - 3
ER -