Description
General description
Full-length human CYSLTR1 cDNA
The cysteinyl leukotrienes, leukotriene C4, leukotriene D4 and leukotriene E4, are arachidonic acid derivatives modified by glutathione, Cys-Gly or Cys. Activated mast cells release cysteinyl leukotrienes, which cause smooth muscle contraction, airway constriction, and vascular permeability. The biological effects of the cysteinyl leukotrienes are mediated by two GPCRs, CysLT1 and CysLT2. A CysLT1 selective antagonist, montelukast, is used clinically in the treatment of asthma (Brink et al., 2003; Evans, 2002). Chemicon′s CysLT1 membrane preparations are crude membrane preparations made from our proprietary stable recombinant cell lines to ensure high-level of GPCR surface expression; thus, they are ideal HTS tools for screening of antagonists of CysLT1 interactions and its ligands. The membrane preparations exhibit a Kd of 0.97nM for [33H]-Leukotriene D4 (LTD4). With 10.0ug/well CysLT1 membrane prep and 1.5 nM [33H]-Leukotriene D4, a greater than 3-fold signal-to-background ratio was obtained.
Application
Human CysLT1 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.
Radioligand binding assay and GTPγS binding
Biochem/physiol Actions
GPCR Class: A
Protein Target: CysLT1
Target Sub-Family: Leukotriene
Features and Benefits
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 50 mM 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: 50 mM Tris, pH7.4, 20 mM CaCl2, 25 mM MgCl2, 10 mM Glycine, 10 mM L-Cysteine, filtered and stored at 4°C.
Radioligand: [3H] LTD4 (Perkin Elmer#: NET-101)
Wash Buffer: 50 mM Tris, pH 7.4, filtered and stored at 4°C.
Physical form
One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 3-fold signal:background with 3H-labeled LTD4 at 1.5 nM.
Liquid in packaging buffer: 50 mM
Preparation Note
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial
