HTS010M | ChemiSCREEN™ Membrane Preparation Recombinant Rehsus Macaque CCR5 Chemokine Receptor

HTS010M
200 units  
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      Overview

      Replacement Information

      Key Spec Table

      SpeciesHost CellsProtein Target
      Rhesus MacaqueChem-1CCR5
      Description
      Catalogue NumberHTS010M
      Trade Name
      • ChemiScreen
      • Chemicon
      DescriptionChemiSCREEN™ Membrane Preparation Recombinant Rehsus Macaque CCR5 Chemokine Receptor
      OverviewFull-length rhesus macaque CCR5 cDNA encoding CCR5
      Background InformationCCR5 is the receptor for CC chemokines MIP-1α, MIP-1β, and RANTES (Raport et al., 1996), and is preferentially expressed on Th1 lymphocytes (Loetscher et al., 1998). CCR5 is a coreceptor for macrophage-tropic HIV, and its ligands potently inhibit HIV replication in human leukocytes (Cocchi et al., 1995). In addition, HIV-infected patients with the nonfunctional CCR5Δ32 allele exhibit delayed onset of AIDS symptoms (Samson et al., 1996), and pharmacological antagonism of CCR5 inhibits HIV-1 infection (Strizki et al., 2001). Preclinical testing of small molecule antagonists of CCR5 has been hampered by low affinity of the compounds to rodent and dog CCR5, but two such compounds, maraviroc and AD101, have been shown to have potent antagonist activity at rhesus macaque CCR5, which differs from human CCR5 by 8 amino acids (Napier et al., 2005; Billick et al., 2004). One antagonist of human CCR5, SCH-C, does not block HIV entry through rhesus macaque CCR5, and one amino acid difference is responsible for the functional difference (Billick et al., 2004). Millipore's rhesus macaque CCR5 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 agonists and antagonists of rCCR5. The membrane preparations exhibit a Kd of 0.32 nM for [125I]-MIP-1α. With 0.25 nM [125I]-MIP-1α, 5 μg/well rCCR5 Membrane Prep typically yields greater than 7-fold signal-to-background ratio.
      References
      Product Information
      FormatMembranes
      PresentationLiquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.
      Packaging method: Membranes protein were adjusted to 1 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80°C
      Applications
      ApplicationRhesus Macaque CCR5 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.
      Key Applications
      • Radioligand binding assay
      • GTPγS binding
      Application NotesRadioligand binding assay and GTPγS binding.
      Biological Information
      Entrez Gene Number
      Gene Symbol
      • CCR5
      • CMKBR5
      GPCR ClassA
      Host CellsChem-1
      Protein TargetCCR5
      Target Sub-FamilyChemokine
      UniProt Number
      Incubation ConditionsRECOMMENDED 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 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: [125I]-MIP-1α (Perkin Elmer#: NEX-298 )

      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 7-fold signal:background with 125I labeled MIP-1α at 0.15 nM
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of CCR5 Receptor membrane prep.
      Product Usage Statements
      10 µg/well 5 µg/well
      Signal:Background 13.6 12.1
      Specific Binding (cpm) 5869 4538


      SPECIFICATIONS: 1 unit = 5 µg membrane preparation
      Bmax for [125I]-MIP-1α binding: 1.9 pmol/mg protein
      Kd for [125I]-MIP-1α binding: ~0.35 nM Usage Statement
      • 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.
      Storage and Shipping Information
      Storage ConditionsMaintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
      Packaging Information
      Material Size200 units
      Transport Information
      Supplemental Information
      Specifications

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      Categories

      Life Science Research > Drug Discovery and Development > GPCR Cell Lines & Assays > GPCR Membrane Preparations
      Life Science Research > Proteins and Enzymes > GPCR Membrane Preparations