Calcium No WashPLUS Assay Principle
The calcium G protein-dependent pathway involves a heterotrimeric (α/β/γ) G-protein containing a GDP molecule (not shown here) bound to the Gα subunit, which holds the trimer together. Upon activation, GDP is exchanged for GTP, leading to the dissociation of the Gβ/Gγ dimer from Gα. Both parts remain anchored to the membrane and become free to act upon their downstream effectors and initiate unique intracellular signaling responses. The activated Gα subunit interacts with and regulates many effector molecules such phospholipase C (PLC) that can ultimately lead to the release of calcium (a second messenger) from the ER.
The Calcium No WashPLUS Detection Kit is calcium mobilization, cell-based assay which uses an esterified (inactive) calcium dye (probe) that penetrates the cell membrane and becomes active once inside the cell. The active form of the dye becomes fluorescent after binding to intracellular calcium. Additive A is added to prevent the dye from being released out from the cell. Ligand binding stimulates GPCR activation, resulting in the release of intracellular calcium stores from the ER, which leads to an increase in fluorescence in the presence of the activated calcium dye.
Perform Rank Order Analysis of Potential Leads
Dose response and rank order potency for calcium mobilization and β-arrestin recruitment assays using the same cell line. The PathHunter CHO-K1 MRGPRX2 β-Arrestin Cell Line (Cat. No. 93-0309C2) was used to analyze both calcium mobilization with the Calcium No WashPLUS detection kit and β-arrestin recruitment. Readouts showing select agonists effect on (A.) calcium mobilization of intracellular calcium and on (B.) β-arrestin recruitment upon binding to MrgprX2 receptors. Both dose response curves are the mean of two replicates and show normalized data calculated from the maximum effect obtained with cortistatin-14. C. Table showing potency and efficacy of agonist relative to cortistatin-14. Results show good agreement with those previously published with respect to rank orders of potency agonists (Grimes et al. 2019) maintained relative to cortistatin-14 used as reference agonist for both assays.