Two-beam fluorescence cross-correlation spectroscopy in an electrophoretic mobility shift assay

Analytical Chemistry
Dale J LeCaptain, Orden Alan Van

Abstract

Two-beam fluorescence cross-correlation spectroscopy (FCCS) was used to resolve the bound and unbound fractions of fluorescently labeled single-stranded DNA (ssDNA) in a ssDNA-protein complex as the analyte solution flowed continuously through an electrophoresis capillary. Cross-correlation of the single molecule fluorescence from two spatially separate excitation laser beams resulted in cross-correlation functions that consisted of well-resolved peaks characteristic of the different electrophoretic flow velocities of the bound and unbound ssDNA. This decoupled the molecular parameters of the bound and unbound ssDNA used to model the cross-correlation function, which enabled the relative concentrations to be determined without prior knowledge of the pure-component cross-correlation functions, as would be required in an analogous autocorrelation analysis. The relative concentrations of the bound and unbound ssDNA were determined by two-beam FCCS within 2-6% precision, even for samples that contained as little as 5% unbound ssDNA, and were consistent with the results obtained by capillary electrophoresis (CE) separation of the same samples. Data sufficient to obtain these results was acquired in 10-15 s per sample. Fluorescently ...Continue Reading

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Citations

Aug 3, 2005·Analytical and Bioanalytical Chemistry·Petra S Dittrich, Andreas Manz
Jun 23, 2009·European Biophysics Journal : EBJ·Gerrit HeuvelmanKarsten Rippe
Sep 18, 2007·Cell Biochemistry and Biophysics·Ling Chin Hwang, Thorsten Wohland
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Jul 21, 2006·The Journal of Physical Chemistry. B·Jaemyeong Jung, Alan Van Orden
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Dec 14, 2007·The Journal of Physical Chemistry. B·Jaemyeong JungAlan Van Orden

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