Functional in vitro Binding Assay [35S]-GTPγS (96-well plate)

[35S]-GTPγS binding, also known as a GTP exchange assay, is a powerful method used to measure the activation of G protein-coupled receptors (GPCRs) by a ligand.

[35S]-GTPγS binding can be used to:

  • Determine if a ligand can activate a GPCR of interest
  • Single concentration screening or dose-response to determine pharmacological values of potency (EC50) & efficacy (EMAX)
  • Determine full, partial, or inverse agonist and antagonist activity
96-well drug plate setup diagram for functional binding assays. The functional binding plate is designed to contain two buffer controls (Column 1 and 11), one agonist control (Column 2), one antagonist control (Column 12) and 2 compounds (Columns 2 - 10). Agonists are subjected to full concentration-response (eight serial drug dilutions are made in quadruplicate) to determine their efficacy and potency. Antagonists are subjected to full concentration-response (eight serial drug dilutions are made in quadruplicate) studies to determine IC50 values against EC50 to EC80 concentrations of a reference agonist. Results are normalized and transformed to percentage values. For agonists, the reference agonist activity at 10 μM is set as 100% and basal activity with buffer as 0%. For antagonists, the basal activity with buffer only is set as 100% inhibition and the activity of the EC80 of the reference agonist as 0% inhibition. For allosteric potentiators, the activity of the EC20 of a reference agonist is set as 0% potentiation.
DPAT Seratonin [35S]GTPyS Functional Assay
96-well drug plate setup diagram for functional binding assays. The functional binding plate is designed to contain two buffer controls (Column 1 and 11), one agonist control (Column 2), one antagonist control (Column 12) and 16 compounds (Columns 2 - 10) at a single concentration. Results are normalized and transformed to percentage values. For agonists, the reference agonist activity at 10 μM is set as 100% and basal activity with buffer as 0%. For antagonists, the basal activity with buffer only is set as 100% inhibition and the activity of the EC80 of the reference agonist as 0% inhibition. For allosteric potentiators, the activity of the EC20 of a reference agonist is set as 0% potentiation. Potential agonist hits are subjected to full concentration-response studies to determine their efficacy and potency. Potential antagonist hits are subjected to full concentration-response studies to determine IC50 values against EC50 to EC80 concentrations of a reference agonist.