Description
There are three subtypes of vasopressin receptor, V1A, V1B, and V2. The V2 receptor is mainly situated in the kidney and is important in free water reabsorption.
Cells expressing the V2 receptor are provided pre-labeled with Terbium, and can be used to conduct receptor binding studies on the aforementioned receptor.
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
Assay principle
Running your V2 receptor binding assay using Tag-lite is as easy as it can gets. 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.
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.
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 labeled cells and their matching fluorescent ligand (L0063RED). Vasopressin was used as reference ligand. Results may vary from one HTRF® compatible reader to another.
