Two-Beam Fluorescence Cross-Correlation Spectroscopy in an Electrophoretic Mobility Shift Assay

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• 作者：Dale J. LeCaptain and Alan Van Orden
• 刊名：Analytical Chemistry
• 出版年：2002
• 出版时间：March 1, 2002
• 年：2002
• 卷：74
• 期：5
• 页码：1171 - 1176
• 全文大小：83K
• 年卷期：v.74,no.5(March 1, 2002)
• ISSN：1520-6882

文摘

Two-beam fluorescence cross-correlation spectroscopy(FCCS) was used to resolve the bound and unboundfractions of fluorescently labeled single-stranded DNA(ssDNA) in a ssDNA-protein complex as the analytesolution flowed continuously through an electrophoresiscapillary. Cross-correlation of the single molecule fluorescence from two spatially separate excitation laserbeams resulted in cross-correlation functions that consisted of well-resolved peaks characteristic of the differentelectrophoretic flow velocities of the bound and unboundssDNA. This decoupled the molecular parameters of thebound and unbound ssDNA used to model the cross-correlation function, which enabled the relative concentrations to be determined without prior knowledge of thepure-component cross-correlation functions, as would berequired in an analogous autocorrelation analysis. Therelative concentrations of the bound and unbound ssDNAwere 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 resultsobtained by capillary electrophoresis (CE) separation ofthe same samples. Data sufficient to obtain these resultswas acquired in 10-15 s per sample. Fluorescentlylabeled poly(dT)₃₉ complexed with the single strandedDNA binding protein of Escherichia coli served as themodel system. The measured dissociation constant of 2.5± 0.9 nM agreed with the literature value for this complexwithin experimental error. The CE/two-beam FCCS experiment described here is part of a family of techniquesthat use single molecule fluorescence detection to resolvedifferent components in an electrophoresis system. Advantages of these methods relative to separations-basedCE include enhanced sensitivity, the potential for higherspeed analyses, elimination of the sample plug injectionstep, and the ability to carry out the analysis in shorterflow channels.