A homogeneous method for measuring the concentration of inositol 1, 4, 5-trisphosphate (IP3) in a sample comprising incubating a protein comprising a Pleckstrin homology domain and a capture moiety with radiolabelled IP3 to produce a complex, capturing said complex on a solid phase that comprises a phosphor and a capture agent that binds the capture moiety, incubating the solid phase where the labelled IP3 is displaced by unlabelled IP3 present in the sample and detecting the amount of bound labelled IP3. Pleckstrin homology domains suitable include those from PLC, dynamin and Brutons tyrosine kinase. Suitable capture moieties/agents include biotin/streptavidin, maltose/MBP and His-tag. The method may be used to determine the effects of test agents on IP3 concentration. Embodiments describe use of a PLC (phospholipase C) Pleckstrin homology domain GST fusion protein where IP3 binding is detected using a scintillation proximity assay (SPA).
Inositol 1, 4, 5 trisphosphate assays