ChemiSCREEN Membrane Preparation Recombinant Human NK₂ Tachykinin Receptor

FUNCTION: SwissProt: P21452 # This is a receptor for the tachykinin neuropeptide substance K (neurokinin A). It is associated with G proteins that activate a phosphatidylinositol-calcium second messenger system.
SIZE: 398 amino acids; 44439 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
SIMILARITY: SwissProt: P21452 ## Belongs to the G-protein coupled receptor 1 family.
MISCELLANEOUS: The rank order of affinity of this receptor to tachykinins is: substance K > neuromedin-K > substance P.

Radioligand binding assay and GTPγS binding

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Packaging method: Membranes protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80oC.

Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.

RECOMMENDED ASSAY CONDITIONS: Membranes are mixed with radioactive ligand and unlabeled competitor (see Figures 1 and 2 for concentrations tested) in binding buffer in a nonbinding 96-well plate, and incubated for 1-2 h. Prior to filtration, an FC 96-well harvest plate (Millipore cat. # MAHF C1H) is coated with 0.33% polyethyleneimine for 30 min, then washed with 50mM Tris, pH 7.4. Binding reaction is transferred to the filter plate, and washed 3 times (1 mL per well per wash) with Wash Buffer. The plate is dried and counted.

Binding buffer: 50mM Tris, pH 7.4, 150mM NaCl, 3mM MnCl₂, 0.1% BSA, containing one Protease Inhibitor Cocktail Tablet (Roche Cat. No. 11 873 580 001) for each 50ml binding buffer.
Radioligand: [¹²⁵I]-NKA (NEX 252)
Wash Buffer: 50mM Tris, pH 7.4, 150mM NaCl, 3mM MnCl₂, 0.1% BSA

One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 10-fold signal:background with ¹²⁵I-labeled NKA at 0.5nM.

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