ChemiSCREEN Membrane Preparation Recombinant Human CysLT ₁ Cysteinyl Leukotriene Receptor

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.

Human CysLT1 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

Radioligand binding assay and GTPγS binding

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.

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

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

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