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

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


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 types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Algorithms*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism*
  • CRISPR-Associated Protein 9
  • DNA / metabolism
  • Endonucleases / metabolism*
  • Fluorescence
  • Protein Binding
  • RNA, Guide, CRISPR-Cas Systems
  • Single Molecule Imaging / methods*


  • Bacterial Proteins
  • IgG Fc-binding protein, Streptococcus
  • DNA
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases