Description
There are three subtypes of vasopressin receptor: V1A, V1B, and V2. The V2 receptor is mainly found in the kidney and is important in free water reabsorption.
This HEK293 stable cell line expresses the V2 receptor fused to a SNAP-Tag. Labeled with Terbium, the cells can be used in a Tag-lite receptor binding protocol.
Tag-lite assays offer many features and applications over traditional receptor/ligand binding assays:
Non-radioactive
Homogeneous and filtration-free
Ready-to-use kits and reagents for binding assays
Does not alter the receptor pharmacology
Peer-reviewed, validated technology
Thorough kinetic studies with true Kd and Ki values, association (Kon) and dissociation (Koff) rate constants
Screening and profiling of biologics and large molecules
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.
Kd and Ki validation
Examples of data obtained using vasopressin V2-R expressing stable cells (HEK) labelled with terbium in Tag-lite buffer (LABMED), and their matching fluorescent ligand (L0063RED). Vasopressin was used as reference ligand. Results may vary from one HTRF® compatible reader to another.
