Step 1 - Receptor labeling

SNAP-tag® is a small fusion tag that covalently interacts with specific substrates. It enables specific covalent labeling of any protein of interest. For more details, see the labeling procedure.

Step 2 - 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 3 - 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 4 - 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.

Examples of data obtained using chemokine CXCR4-R expressing stable cells (HEK) labelled with terbium in Tag-lite buffer (LABMED), and their matching fluorescent ligand (L0012RED). AMD-3100 was used as reference ligand. Results may vary from one HTRF® compatible reader to another.