A direct, homogenous antagonist assay can be run on the same cell line that was used for an agonist assay. Again a very simple (no wash) protocol can be used:
- Add agonist, red fluorogen, plus test compound (antagonist)
- allow time for response and add green fluorogen
- Read
If the test compound acts as an antagonist, then all of the receptor will remain on the surface and it will all end up with the green fluorogen. If some of the receptor did internalize it would be red, as the green fluorogen would not be able to exchange with the red in the endosome. The images below show two examples of the results of increasing concentrations of antagonist using this assay.
In both of these cases the antagonist completely blocks the agonist response (No red signal) at about 100 nMolar. SpectraGenetics has tagged more than 150 GPCRs and has validated more than 30 cell lines.