Description
General description
BLT1 receptor is a Gi-coupled GPCR that binds primarily to the eicosanoid leukotriene B4. Macrophages, leukocytes and eosinophils express BLT1, and in vivo, BLT1 appears to mediate leukocyte migration and inflammation (reviewed in Brink et al., 2003). Chemicon′s BLT1 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 BLT1 interactions with Leukotriene B4. The membrane preparations exhibit a Kd of 1.3 nM for [3H]-Leukotriene B4. With 5.0 μg/well BLT1 Membrane Prep and 1 nM [3H]-Leukotriene B4, a signal-to-background ratio of approximately 3-fold is obtained.
Human BLT1
Application
Human BLT1 GPCR membrane preparation for Radioligand binding Assays & GTPgammaS binding.
Radioligand binding assay, and GTPgammaS binding.
Biochem/physiol Actions
GPCR Class: A
Protein Target: BLT1
Target Sub-Family: Leukotriene
Features and Benefits
Inucbation Conditions
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, a GF/C 96-well filter plate is coated with 0.33% polyethyleneimine for 30 min, then washed with 50mM HEPES, pH 7.4, 0.5% BSA. 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 Hepes, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.2% BSA, filtered and stored at 4°C
Radioligand: [3H] Luekotriene (Perkin Elmer# NEX378)
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, filtered and stored at 4°C.
One package contains enough membranes for at least 200 assays (units), where a unit is the amount of membrane that will yield greater than 2.5-fold signal:background or 500 cpm with 3H-labeled Leukotriene B4 at 1 nM
Physical form
Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.
Packaging method: Membranes protein were adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80°C.
Preparation Note
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
Analysis Note
SPECIFICATIONS:
Signal:background and specific binding values obtained in a competition binding assay with varying amounts of Motilin Receptor membrane prep: 5 µg/well10 µg/well
Signal:Background 75 65
Specific Binding (cpm) 46348 54457
1 unit = 5µg membrane preparation
Bmax: 5.6 pmol/mg
Kd: 0.43 nM
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 uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
