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PrecisION™ MembranePreparation Recombinant hERG

市场价：
10.00
促销价：
¥0.00

货号：CYL4039

品牌：Millipore 密理博

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Replacement Information

重要规格表

Species Host Cells Protein Target
Human 293 cell line hERG
Description
Catalogue Number CYL4039
Trade Name
  • PrecisION
Description PrecisION™ hERG ion channel membrane preparation
Overview Human ERG
Background Information The 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
Format Membranes
Presentation Liquid 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.
Applications
Application Human ERG ion channel membrane preparation for Radioligand binding Assays.
Application Notes Radioligand binding assay
Biological Information
Entrez Gene Number
Host Cells 293 cell line
Protein Target hERG
Target Sub-Family Potassium
Incubation 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, 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 PatchExpress Literature value by Patch Clamp
Astemizole 3.4 ± 0.7 3.3 ± 0.7 4.6 ± 0.003 ND 0.5
Dofetilide 5.8 ± 2.4 28 ± 6 ND ND 15.3 ± 2.5
E-4031 6.1 ± 0.8 58 ± 5 115 ± 0.02 144 ± 44 14
Pimozide 18.8 ± 2.3 14 ± 4 ND ND 18
Cisapride 104.9 ± 30.9 123 ± 25 75.6 ± 0.02 5 ± 1 44.5 ± 10.6
Haloperidol 241.4 ± 21.4 234 ± 20 ND ND 63
Risperidone > 4000 4302 ± 319 ND ND 167
Verapimil > 4000 3902 ± 529 1332 ± 120 3100 ± 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 Conditions Maintain frozen at –70°C up to expiration date indicated on the label. Do not freeze and thaw.
Packaging Information
Material Size 200 units
Transport Information
Supplemental Information
Specifications

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