AlphaScreen-based CaM interaction assay.
Optical stimulation of phthalocyanine compounds contained in the AlphaScreen donor beads excites ambient oxygen molecules into the metastable singlet state. The energy conveyed by the molecule is converted into light emission by the acceptor bead. Owing to the limited lifespan of singlet oxygen in solution, the AlphaScreen signal emission is highly dependent on the distance between the two beads. In this study, AlphaScreen donor and acceptor beads were modified for the detection of CaM/TRPV4 or interdomain interactions. Streptavidin-coated donor and anti-Flag acceptor beads were used to attach the proteins to the bead surface.