CYL4039 PrecisION™ hERG ion channel membrane preparation

CYL4039
200 units  
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      Overview

      Replacement Information

      Key Spec Table

      SpeciesHost CellsProtein Target
      Human 293 cell line hERG
      Description
      Catalogue NumberCYL4039
      Trade Name
      • PrecisION
      DescriptionPrecisION™ hERG ion channel membrane preparation
      OverviewHuman ERG
      Background InformationThe human ether-a-go-go related gene (hERG) is a potassium ion channel which is essential for normal cardiac repolarization. In drug screening models, the hERG K+ channel has been indicated to inhibit a wide variety of compounds, and its blockage can lead to cardiac QT interval prolongation and life threatening arrhythmias (Murphy et al. 2006). Cardiac safety relating to IKr K+ channels has become a major concern of regulatory agencies, as hERG channel inhibition has been identified as the firmest link to QT prolongation (Chiu et al. 2004). Millipore's hERG membrane preparations are crude membrane preparations made from HEK293 stable recombinant cell lines (Millipore cat. # CYL3039), which are ideal HTS tools for screening antagonists against the hERG channel. The membrane preparations exhibit a Kd of 5.2 nM for [3H]-Astemizole. With 10 μg/well hERG Membrane Prep and 3.0 nM [3H]-Astemizole, a greater than 4-fold signal-to-background ratio was obtained.
      References
      Product Information
      FormatMembranes
      PresentationLiquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
      Packaging method: Membrane protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80°C.
      Quality LevelMQ100
      Applications
      ApplicationHuman ERG ion channel membrane preparation for Radioligand binding Assays.
      Application NotesRadioligand binding assay
      Biological Information
      Entrez Gene Number
      Host Cells293 cell line
      Protein TargethERG
      Target Sub-FamilyPotassium
      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 FB 96-well harvest plate (Millipore cat. # MAHF B1H) is coated with 0.33% polyethyleneimine for 30 min, then washed with 25 mM Tris, pH 7.4, 130 mM NaCl, 5 mM KCl, 0.8 mM CaCl2, 0.1% BSA. Binding reaction is transferred to the filter plate, and washed 6 times (250μL per well per wash) with Wash Buffer. The plate is dried and counted.

      Binding buffer: 10 mM Hepes, pH 7.4, 130 mM NaCl, 5 mM KCl, 0.8 mM MgCl2, 1 mM NaEGTA, 10 mM glucose, 0.1% BSA, filtered and stored at 4°C

      Radioligand: [3H]-Astemizole (Perkin Elmer # NET-1140)

      Wash Buffer: 25 mM Tris, pH 7.4, 130 mM NaCl, 5 mM KCl, 0.8 mM MgCl2, 0.05 mM CaCl2, 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 4-fold signal: background with 3H-labeled Astemizole.
      Cell Line Type
      • GPCR Cell Lines
      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 for the hERG membrane prep.
      Product Usage Statements
      10 µg/well
      Signal:Background 4.6
      Specific Binding (cpm) 435


      SPECIFICATIONS: 1 unit = 10 μg membrane preparation with [3H]-astemizole
      Bmax with [3H]-astemizole: 6.7 pmol/mg
      Kd for [3H]-astemizole: 5.2 nM

      Table 2. Rank order comparison of various small molecule hERG inhibitors using [3H]-astemizole binding assay and E-Phys assays:
      Binding Ki (nM) with hERG membrane preps Electrophysiology IC50 (nM) with hERG cell lines
      Millipore CYL4039M Literature value* Millipore CYL4039 by IonWorks PPC Millipore CYL4039 by PatchExpressLiterature value by Patch Clamp
      Astemizole 3.4 ± 0.7 3.3 ± 0.7 4.6 ± 0.003 ND 0.5
      Dofetilide 5.8 ± 2.428 ± 6NDND15.3 ± 2.5
      E-4031 6.1 ± 0.8 58 ± 5115 ± 0.02 144 ± 44 14
      Pimozide 18.8 ± 2.3 14 ± 4NDND18
      Cisapride 104.9 ± 30.9 123 ± 25 75.6 ± 0.02 5 ± 1 44.5 ± 10.6
      Haloperidol 241.4 ± 21.4 234 ± 20 NDND63
      Risperidone > 4000 4302 ± 319NDND167
      Verapimil > 4000 3902 ± 529 1332 ± 1203100 ± 400 830

      *Data obtained from Chiu et al., 2004


      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 up to expiration date indicated on the label. Do not freeze and thaw.
      Packaging Information
      Material Size200 units
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      PrecisION™ hERG ion channel membrane preparation MSDS

      Title

      Safety Data Sheet (SDS) 

      PrecisION™ hERG ion channel membrane preparation Certificates of Analysis

      TitleLot Number
      PRECISIONTM MEMBRANE PREPARATION RECOMBINANT HERG POTASSIUM ION CHANNEL - 21950522195052
      PRECISIONTM MEMBRANE PREPARATION RECOMBINANT HERG POTASSIUM ION CHANNEL - 22116252211625

      Brochure

      Title
      Product Selection Guide - Antibodies, small molecule inhibitors, kits, assays and proteins for signaling research.

      Data Sheet

      Title
      PRECISIONTM MEMBRANE PREPARATION RECOMBINANT hERG POTASSIUM ION CHANNEL
      Precision Membrane Preparation Recombinant hERG Potassium Ion Channel

      Posters

      Title
      Assessment Of hERG Blockade By Radioligand Binding Assay With hERG Membrane Preparations
      Development and Validation of Automated Electrophysiology Assays for Voltage-gated Sodium Channels

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      Categories

      Life Science Research > Cell Culture and Systems > Cell Lines > Ion Channel Cell Lines
      Life Science Research > Drug Discovery and Development > Ion Channel Cell Lines & Assays > Ion Channel Cell Lines
      Life Science Research > Drug Discovery and Development > Ion Channel Cell Lines & Assays > Ion Channel Membrane Preparations
      Life Science Research > Proteins and Enzymes > Ion Channel Membrane Preparations