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. 2009 Sep 16;37(21):e142. doi: 10.1093/nar/gkp733

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© The Author(s) 2009. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 6. — Microfluidic 3WJ RNA–S15 protein binding kinetics. (A) Binding of the fluorescently labeled protein S15-Alexa594 to the doubly labeled 3WJ RNA results in efficient FRET to S15-Alexa594 (24). (B) Time trace of a 5-nl scale binding reaction prepared by metering 3WJ RNA into a reaction ring filled with serially diluted S15. Mixing creates damped oscillations for the first 10 s, after which the peristaltic pumps are locked closed and fluorescence changes due to FRET. (C) Eleven reactions differing in the initial concentration of S15 were run in quadruplicate and apparent binding constants (K_app) were recovered by fitting FRET donor intensity to mono-exponential decays. (D) Fitting the averaged K_app to S15 concentration (Equation 1) recovers the bimolecular on-rate K_on and unimolecular off-rate K_off. The ratio of the rates yields an affinity (K_D = 8 ± 2 nm) which agrees with the affinity obtained from 200 μl scale assays with the same materials (10.7 ± 0.8nM, data not shown) and literature values (23,26).