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

Boyang Hua, Yanbo Wang, Seongjin Park, Kyu Young Han, Digvijay Singh, Jin H. Kim, Wei Cheng, Taekjip Ha

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)1572-1576
Number of pages5
JournalBiochemistry
Volume57
Issue number10
DOIs
StatePublished - Mar 13 2018

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Artifacts
Assays
Immunoglobulin G
Fluorescence
RNA
Molecules
DNA
Experiments
Streptococcus IgG Fc-binding protein

ASJC Scopus subject areas

  • Biochemistry

Cite this

The Single-Molecule Centroid Localization Algorithm Improves the Accuracy of Fluorescence Binding Assays. / Hua, Boyang; Wang, Yanbo; Park, Seongjin; Han, Kyu Young; Singh, Digvijay; Kim, Jin H.; Cheng, Wei; Ha, Taekjip.

In: Biochemistry, Vol. 57, No. 10, 13.03.2018, p. 1572-1576.

Research output: Contribution to journalArticle

Hua, Boyang ; Wang, Yanbo ; Park, Seongjin ; Han, Kyu Young ; Singh, Digvijay ; Kim, Jin H. ; Cheng, Wei ; Ha, Taekjip. / The Single-Molecule Centroid Localization Algorithm Improves the Accuracy of Fluorescence Binding Assays. In: Biochemistry. 2018 ; Vol. 57, No. 10. pp. 1572-1576.
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