Step 1 - Plasmid transfection

Use standard transfection techniques (refer to transient transfection protocol) to transiently express the SNAP-GPCR of interest in your cell line.

Step 2 - Receptor labeling

SNAP-tag® is a small fusion tag that covalently interacts with specific substrates. It allows specific and covalent labeling of any protein of interest (refer to labeling procedure). Cells are provided unlabeled and need to be labeled with Lumi4-Terbium prior to running a binding assay. Labeling reagents are available from the Revvity catalog in 4 different sizes.

Step 3 - Understand the assay principle

Running a receptor binding assay using Tag-lite is as easy as it can get. Simply dispense 10 µL of labeled cells into each well, followed by 5 µL of labeled ligand and 5 µL of the compound you wish to test. Like all HTRF assays, Tag-lite assays do not require any washing steps. A diagram of the procedure to be followed is given on the right.

Step 4- Saturation binding (KD)

A saturation binding assay measures total and non-specific binding for increasing concentrations of ligand under equilibrium conditions. To perform the assay, the fluorescent ligand is titrated into a solution containing a fixed amount of labeled cells and then incubated to equilibrium. The HTRF ratio obtained from this titration is the total binding.

Step 5 - Competitive binding (KI)

A competitive binding assay is performed to measure the dissociation constant, Ki. To perform the assay, the compound is titrated into a solution containing a fixed concentration of fluorescent ligand and a fixed amount of cells.