Competitive Fluorescent Ligand Assay for Inositol 1,4,5-Trisphosphate

We present a novel method, termed competitive fluorescent ligand assay for inositol 1,4,5-trisphosphate (CFLA-IP₃), to measure inositol 1,4,5-trisphosphate (IP₃). This method is based on fluorescence resonance energy transfer (FRET) between two fluorescent molecules, a fluorescent IP₃-binding protein and its fluorescent ligand. Binding of these fluorescent molecules generates a FRET signal, and the IP₃-dependent decrease in the FRET signal due to displacement of the fluorescent ligand is detected by fluorescence microscopy.