GTPγS Assay Protocol

In agonist-induced activation of G-protein coupled receptors, GTP binding to the alpha subunit of heterotrimeric GTP binding proteins is an early event. GTPγS binding tests are similar to radioligand binding assays in that they use membrane preparations. Such experiments are performed with [³⁵S] guanosine-5'-O-(3-thio) triphosphate, which is a radioactive ligand with a high affinity for G-protein subunits and is very resistant to the subunits' natural GTPase activity, allowing it to remain bound for long enough to allow radioactivity counting.

GTPγS Assay Protocol

Assay using WGA SPA beads

1. Incubate membranes, GTPγ³⁵S, and compounds evaluated in 200 μL/well for 30-60 minutes at room temperature.
2. Pour 50 μL of suspended WGA beads into each well.
3. Cover plates and incubate at room temperature for one hour or more.
4. Count plates after centrifugation at 200g.

Antibody capture assay

1. Prepare the membranes as directed for the entire membrane assay.
2. Pour 20 μL of 3% NP40 into each well and incubate for 15 minutes.
3. Add 20 μL of primary antibody to each well and incubate for 15 minutes.
4. Pour 50 μL of anti-rabbit or anti-mouse SPA beads into each well.
5. Seal and incubate for three hours, then centrifuge and count as directed above