Quantitative affinity determination by fluorescence anisotropy measurements of individual nanoliter droplets

File Description SizeFormat 
acs%2Eanalchem%2E6b02528.pdfPublished version2.26 MBAdobe PDFView/Open
Title: Quantitative affinity determination by fluorescence anisotropy measurements of individual nanoliter droplets
Authors: Gielen, F
Butz, M
Rees, EJ
Erdelyi, M
Moschetti, T
Hyvonen, M
Edel, JB
Kaminski, CF
Hollfelder, F
Item Type: Journal Article
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.
Issue Date: 3-Jan-2017
Date of Acceptance: 1-Dec-2016
URI: http://hdl.handle.net/10044/1/45098
DOI: https://dx.doi.org/10.1021/acs.analchem.6b02528
ISSN: 0003-2700
Publisher: American Chemical Society
Start Page: 1092
End Page: 1101
Journal / Book Title: Analytical Chemistry
Volume: 89
Issue: 2
Copyright Statement: © 2017 American Chemical Society. This is an open access article published under a Creative Commons Attribution License (CC-BY http://pubs.acs.org/page/policy/authorchoice_ccby_termsofuse.html), which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
Keywords: Science & Technology
Physical Sciences
Chemistry, Analytical
Chemistry
PROTEIN-PROTEIN INTERACTIONS
CELL-LYSATE ASSAYS
DIRECTED EVOLUTION
HUMAN RAD51
POLARIZATION
MICROFLUIDICS
COMPARTMENTS
INHIBITORS
PLATFORM
MICRODROPLETS
Analytical Chemistry
0301 Analytical Chemistry
0904 Chemical Engineering
0399 Other Chemical Sciences
Publication Status: Published
Appears in Collections:Chemistry
Faculty of Natural Sciences



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Creative Commonsx