Screening for protein kinase C ligands using fluorescence resonance energy transfer.

Zn(II)-Coordinated Quantum Dot-FRET Nanosensors for the Detection of Protein Kinase Activity

We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coo...

Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noi...

Corrigendum: Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

Expanding TranscreenerTM Assay Technology: ADP Detection using TR-FRET

Introduction The TranscreenerTM KINASE TR-FRET Assay is a competitive HTS assay that measures ADP, the invariant product of all kinase reactions. Previously we developed a monoclonal antibody and a fluorescent tracer that allow detection of ADP with >100-fold selectivity over ATP using a fluorescence polarization (FP) readout. To offer detection options to the HTS community, we formatted the Tr...

Fluorescence Resonance Energy Transfer in Quantum Dot-Protein Kinase Assemblies

In search of viable strategies to identify selective inhibitors of protein kinases, we have designed a binding assay to probe the interactions of human phosphoinositide-dependent protein kinase-1 (PDK1) with potential ligands. Our protocol is based on fluorescence resonance energy transfer (FRET) between semiconductor quantum dots (QDs) and organic dyes. Specifically, we have expressed and puri...