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Quantitative Affinity Determination by Fluorescence Anisotropy Measurements of Individual Nanoliter Droplets.

Authors
  • Gielen, Fabrice1, 2
  • Butz, Maren1
  • Rees, Eric J3
  • Erdelyi, Miklos3, 4
  • Moschetti, Tommaso1
  • Hyvönen, Marko1
  • Edel, Joshua B5
  • Kaminski, Clemens F3
  • Hollfelder, Florian1
  • 1 Department of Biochemistry, University of Cambridge , 80 Tennis Court Road, Cambridge, CB2 1GA, United Kingdom. , (United Kingdom)
  • 2 Living Systems Institute, University of Exeter , Stocker Road, Exeter, EX4 4QD, United Kingdom. , (United Kingdom)
  • 3 Department of Chemical Engineering and Biotechnology, New Museums Site , Pembroke Street, Cambridge, CB2 3RA, United Kingdom. , (United Kingdom)
  • 4 Department of Optics and Quantum Electronics, University of Szeged , Dom ter 9, Szeged, Hungary. , (Hungary)
  • 5 Department of Chemistry, Imperial College London , South Kensington, London, SW7 2AZ, United Kingdom. , (United Kingdom)
Type
Published Article
Journal
Analytical Chemistry
Publisher
American Chemical Society
Publication Date
Jan 17, 2017
Volume
89
Issue
2
Pages
1092–1101
Identifiers
DOI: 10.1021/acs.analchem.6b02528
PMID: 28192993
Source
Medline
Language
English
License
Unknown

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, Kd values 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.

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