title: Quantitative Affinity Determination by Fluorescence Anisotropy Measurements of Individual Nanoliter Droplets.
abstract: Fluorescence anisotropy measurements of reagents compartmentalized into individual nanoliter droplets are shown to yield high-resolution binding curves from which precise dissociation constants (Kd) for protein-peptide interactions can be inferred. With the current platform, four titrations can be obtained per minute (based on ∼100 data points each), with stoichiometries spanning more than 2 orders of magnitude and requiring only tens of microliters of reagents. In addition to affinity measurements with purified components, Kdvalues for unpurified proteins in crude cell lysates can be obtained without prior knowledge of the concentration of the expressed protein, so that protein purification can be avoided. Finally, we show how a competition assay can be set up to perform focused library screens, so that compound labeling is not required anymore. These data demonstrate the utility of droplet compartments for the quantitative characterization of biomolecular interactions and establish fluorescence anisotropy imaging as a quantitative technique in a miniaturized droplet format, which is shown to be as reliable as its macroscopic test tube equivalent.
authors.affiliation: Department of Biochemistry, University of Cambridge , 80 Tennis Court Road, Cambridge, CB2 1GA, United Kingdom.|Living Systems Institute, University of Exeter , Stocker Road, Exeter, EX4 4QD, United Kingdom.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Gielen
authors.affiliation: Department of Biochemistry, University of Cambridge , 80 Tennis Court Road, Cambridge, CB2 1GA, United Kingdom.
authors.forename: Maren
authors.initials: M
authors.lastname: Butz
authors.affiliation: Department of Chemical Engineering and Biotechnology, New Museums Site , Pembroke Street, Cambridge, CB2 3RA, United Kingdom.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Rees
authors.affiliation: Department of Chemical Engineering and Biotechnology, New Museums Site , Pembroke Street, Cambridge, CB2 3RA, United Kingdom.|Department of Optics and Quantum Electronics, University of Szeged , Dom ter 9, Szeged, Hungary.
authors.forename: Miklos
authors.initials: M
authors.lastname: Erdelyi
authors.affiliation: Department of Biochemistry, University of Cambridge , 80 Tennis Court Road, Cambridge, CB2 1GA, United Kingdom.
authors.forename: Tommaso
authors.initials: T
authors.lastname: Moschetti
authors.affiliation: Department of Biochemistry, University of Cambridge , 80 Tennis Court Road, Cambridge, CB2 1GA, United Kingdom.
authors.forename: Marko
authors.initials: M
authors.lastname: Hyvönen
authors.affiliation: Department of Chemistry, Imperial College London , South Kensington, London, SW7 2AZ, United Kingdom.
authors.forename: Joshua B
authors.initials: JB
authors.lastname: Edel
authors.affiliation: Department of Chemical Engineering and Biotechnology, New Museums Site , Pembroke Street, Cambridge, CB2 3RA, United Kingdom.
authors.forename: Clemens F
authors.initials: CF
authors.lastname: Kaminski
authors.affiliation: Department of Biochemistry, University of Cambridge , 80 Tennis Court Road, Cambridge, CB2 1GA, United Kingdom.
authors.forename: Florian
authors.identifier: 0000-0002-1367-6312
authors.initials: F
authors.lastname: Hollfelder