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Biochemistry. 2018 Mar 13;57(10):1572-1576. doi: 10.1021/acs.biochem.7b01293. Epub 2018 Feb 28.

The Single-Molecule Centroid Localization Algorithm Improves the Accuracy of Fluorescence Binding Assays.

Author information

1
Department of Biophysics and Biophysical Chemistry , Johns Hopkins School of Medicine , Baltimore , Maryland 21205 , United States.
2
Department of Physics , University of Illinois at Urbana-Champaign , Urbana , Illinois 61801 , United States.
3
College of Pharmacy , University of Michigan , Ann Arbor , Michigan 48109 , United States.
4
T. C. Jenkins Department of Biophysics , Johns Hopkins University , Baltimore , Maryland 21218 , United States.
5
Howard Hughes Medical Institute , Baltimore , Maryland 21205 , United States.

Abstract

Here, we demonstrate that the use of the single-molecule centroid localization algorithm can improve the accuracy of fluorescence binding assays. Two major artifacts in this type of assay, i.e., nonspecific binding events and optically overlapping receptors, can be detected and corrected during analysis. The effectiveness of our method was confirmed by measuring two weak biomolecular interactions, the interaction between the B1 domain of streptococcal protein G and immunoglobulin G and the interaction between double-stranded DNA and the Cas9-RNA complex with limited sequence matches. This analysis routine requires little modification to common experimental protocols, making it readily applicable to existing data and future experiments.

PMID:
29457977
PMCID:
PMC6149537
[Available on 2019-03-13]
DOI:
10.1021/acs.biochem.7b01293
[Indexed for MEDLINE]

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