Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells.

ABSTRACT: We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell auto

SUBMITTER: Schaaf TM 

PROVIDER: S-EPMC5506495 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature