Principle of time-resolved Scintillation Proximity Assay (SPA) to quantify stability of high-affinity radio-ligand binding to GPCRs.
A) Receptors are immobilized on the SPA beads. When radioligands bind to immobilized receptors, energy from the radioisotope decay is efficiently transferred to the scintillator contained in the beads, thus generating a detectable light signal. If the buffer, including detergent, causes the loss of receptor affinity to radiolabeled ligand, the signal will start to decrease irreversibly. The decay of high-affinity ligand binding to receptors can be measured when the same assay is counted repeatedly over time. B) Comparison of desalting gravity flow separation columns (blue circles) and SPA (black circles) to measure decrease in binding of [3H]-DPN to KOR over time at room temperature. KOR was solubilized in 1%/0.2% DDM/CHS (w/v) and diluted in the assay to 0.1%/0.02% DDM/CHS (w/v). Specific binding was normalized to initial binding. As control of assay stability, the signal of bead-immobilized [3H]-biotin was measured in parallel (red circles). Error bars are standard error of the mean from four (gravity flow columns) or three (SPA) independent experiments each done in triplicates. To quantify the stability of ligand binding, data was fitted to an exponential decay function (solid and dashed lines).
