Agonist-stimulated [35S]GTPγS autoradiography: optimization for high sensitivity

Abstract The receptor-stimulated accumulation of [ 35 S]GTPγS provides a measure of functional coupling of G proteins with receptors. Sensitivity for autoradiographic visualization of [ 35 S]GTPγS binding was improved two- to threefold in rat brain sections by optimizing assay conditions. Non-specific (NSB), basal and agonist-stimulated [ 35 S]GTPγS binding were measured, using methadone, 5-carboxamidotryptamine and epinephrine for μ-opiate receptors, 5-HT 1A receptors and α 2 -adrenoceptors. Basal and NSB were low in glycylglycine buffer compared to Tris or HEPES buffers, and agonist-stimulated [ 35 S]GTPγS binding was more easily observed. Further optimization using glycylglycine buffer found increased signal-to-noise ratio with inclusion of dithiothrietol, increased [ 35 S]GTPγS incubation time (2–4 h) and guanosine 5′-diphosphate (GDP) preincubation (20–30 min), and use of [ 35 S]GTPγS at 0.1 nM. Improved sensitivity was due to decreased NSB and basal [ 35 S]GTPγS binding and agonist-stimulated binding were similarly affected for each receptor system. The assay conditions described should extend the use of agonist-stimulated [ 35 S]GTPγS autoradiography to receptors, which produce low levels of [ 35 S]GTPγS binding and to the measurement of changes in receptor–G protein coupling.