Ligand Binding Assay Protocol
The receptor binding assay can be used to characterize the interaction between the receptor and its ligands in great detail. The receptor binding assay is a cell-free approach for screening GPCRs that does not require downstream signaling from the receptor.
Binding Assay Protocol
Binding assays are carried out in a total volume of 200 μL under the following conditions:
-
Membrane dilution:
7.45 mL assay buffer + 0.05 mL membranes (1:150 dilution) -
Incubation:
25 μL incubation buffer or 10 M final ligand for non-specific binding incubation
Assay for competition binding: 25 μL of decreasing amounts of reference substances
25 μL of radioligand at the desired concentration Membranes diluted in 150 μL -
Incubation time:
30 minutes at 30°C -
Filtration:
Aspirate and wash 9 × 500 μL over GF/C filter with ice cold wash buffer (presoaked in 0.5% BSA).
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