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

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.

Publication Date

3-13-2018

Publication Title

Biochemistry

Volume

57

Issue

10

Number of Pages

1572-1576

Document Type

Article

Personal Identifier

scopus

DOI Link

https://doi.org/10.1021/acs.biochem.7b01293

Socpus ID

85043702664 (Scopus)

Source API URL

https://api.elsevier.com/content/abstract/scopus_id/85043702664

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