Abstract
Life on the molecular scale is based on a complex interplay of biomolecules under which the ability of binding is crucial. Fluorescence based two-color coincidence detection (TCCD) is commonly used to characterize molecular binding, but suffers from an underestimation of coincident events. Here, we introduce a brightness-gated TCCD which overcomes this limitation and benchmark our approach with two custom-made calibration samples. Applied to a cell-free protein synthesis assay, brightness-gated TCCD unraveled a previously disregarded mode of translation initiation in bacteria.
Highlights
Life on the molecular scale is based on a complex interplay of biomolecules under which the ability of binding is crucial
Bimolecular binding of freely diffusing fluorescent molecules is frequently studied by Förster resonance energy transfer (FRET)[12,13] or fluorescence cross-correlation spectroscopy (FCCS)[14]
The Brightness-gated two-color coincidence detection (BTCCD) approach utilizes two different thresholds for the fluorescence signal of each color: the burst threshold is used to discriminate a single-molecule event against the background[24] and the brightness threshold selects bursts that exceed a certain brightness
Summary
Life on the molecular scale is based on a complex interplay of biomolecules under which the ability of binding is crucial. Fluorescence based two-color coincidence detection (TCCD) is commonly used to characterize molecular binding, but suffers from an underestimation of coincident events. Numerous cellular processes, including biochemical signaling or the assembly of higher order complexes such as ribosomes, are regulated through the binding affinities of several interaction partners[1,2,3]. These are determined by biochemical/biophysical techniques like electrophoresis[4], surface plasmon resonance[5], or isothermal titration calorimetry[6]. Two-color coincidence detection (TCCD)[16,17,18] is probably the most straightforward approach to quantify the fraction of bound and unbound molecules in an ensemble of diffusing molecules
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
