FRET-based screening assay using small-molecule photoluminescent probes in lysate of cells overexpressing RFP-fused protein kinases

Anal Biochem. 2015 Jul 15;481:10-7. doi: 10.1016/j.ab.2015.04.009. Epub 2015 Apr 10.

Abstract

An assay was developed for the characterization of protein kinase inhibitors in lysates of mammalian cells based on the measurement of FRET between overexpressed red fluorescent protein (TagRFP)-fused protein kinases (PKs) and luminophore-labeled small-molecule inhibitors (ARC-Photo probes). Two types of the assay, one using TagRFP as the photoluminescence donor together with ARC-Photo probes containing a red fluorophore dye as acceptor, and the other using TagRFP as the acceptor fluorophore in combination with a terbium cryptate-based long-lifetime photoluminescence donor, were used for FRET-based measurements in lysates of the cells overexpressing TagRFP-fused PKs. The second variant of the assay enabled the performance of the measurements under time-resolved conditions that led to substantially higher values of the signal/background ratio and further improved the reliability of the assay.

Keywords: Cell lysate; FRET; Photoluminescent probes; Protein kinases; Red fluorescent protein; TR FRET.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cloning, Molecular
  • Drug Evaluation, Preclinical / methods*
  • Fluorescence Resonance Energy Transfer / methods*
  • HeLa Cells
  • Humans
  • Luminescent Agents / chemistry
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Mice
  • NIH 3T3 Cells
  • Protein Kinase Inhibitors / chemistry
  • Protein Kinase Inhibitors / pharmacology*
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Up-Regulation

Substances

  • Luminescent Agents
  • Luminescent Proteins
  • Protein Kinase Inhibitors
  • Recombinant Fusion Proteins
  • red fluorescent protein
  • Protein Kinases