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ChemiScreen™ Membrane Prep Human Recombinant A3

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货号：HTS052M

品牌：Millipore 密理博

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Replacement Information

重要规格表

Species Host Cells Protein Target
Human Chem-3 A3
Description
Catalogue Number HTS052M
Brand Family Chemicon®
Trade Name
  • ChemiScreen
  • Chemicon
Description ChemiScreen™ A3 Membrane Preparation
Overview Human A3
Background Information Extracellular adenosine mediates a multitude of biological effects, including wakefulness, antiarrythmia, bronchoconstriction and response to ischemia and oxidative stress. A family of four GPCR adenosine receptors, A1, A2A, A2B and A3, is responsible for these effects (Fredholm et al., 2001). A3, which couples to Gi/o, is expressed in mast cells along with A2B. Mice lacking A3 display reduced mast cell degranulation and bronchoconstriction in response to adenosine (Tilley et al., 2003; Zhong et al., 2003). Chemicon's cloned human A3-expressing cell line is made in the Chem-3 host, which supports high levels of recombinant A3 expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for agonists and antagonists at A3.

Chemicon's A3 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 A3 interactions with its ligands. The membrane preparations exhibit a Kd of 4.7 nM for [125I]-AB-MECA. With 5 mg/well A3 Membrane Prep and 5nM [125I]-AB-MECA, a greater than 3-fold signal-to-background ratio is obtained.
References
Product Information
Format Membranes
Presentation 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.
Applications
Application Human A3 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.
Key Applications
  • Radioligand binding assay
  • GTPγS binding
Application Notes Radioligand binding assay and GTPγS binding.
Biological Information
Concentration 1 mL, 1.0 mg/mL
Entrez Gene Number
Entrez Gene Summary This gene encodes a protein that belongs to the family of adenosine receptors, which are G-protein-coupled receptors that are involved in a variety of intracellular signaling pathways and physiological functions. The receptor encoded by this gene mediates a sustained cardioprotective function during cardiac ischemia, it is involved in the inhibition of neutrophil degranulation in neutrophil-mediated tissue injury, it has been implicated in both neuroprotective and neurodegenerative effects, and it may also mediate both cell proliferation and cell death. Multiple transcript variants encoding different isoforms have been found for this gene.
Gene Symbol
  • ADORA3
  • AD026
  • bA552M11.5
  • ORFNames=UNQ1931/PRO4406
  • RP11-552M11.7
  • A3AR
GPCR Class A
Host Cells Chem-3
Protein Target A3
Target Sub-Family Adenosine
UniProt Number
UniProt Summary FUNCTION: SwissProt: P33765 # Receptor for adenosine. The activity of this receptor is mediated by G proteins which inhibits adenylyl cyclase. Possible role in reproduction.
SIZE: 318 amino acids; 36185 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
PTM: Phosphorylation on Ser-317 may be crucial for rapid desensitization (By similarity).
SIMILARITY: SwissProt: P33765 ## Belongs to the G-protein coupled receptor 1 family.
Incubation 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, and 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, pH 7.4, 10 mM MgCl2, 1 mM EDTA, filtered and stored at 4°C

Radioligand: [125I]-AB-MECA (Perkin Elmer# :NEX312)

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 a unit is the amount of membrane that will yield greater than 3-fold signal:background with 125I-labeled AB-MECA at 0.2 nM
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Table 1. 3

Signal:background and specific binding values obtained in a competition binding assay with A membrane preparation:
Product Usage Statements
10 µg/well 5 µg/well
Signal:Background 3.7 4.0
Specific Binding (cpm) 1740 1708


SPECIFICATIONS:
1 unit = 5 μg
Bmax: 2.03 pmol/mg
Kd: 4.7 nM

Table 2. Summary of adenosine receptor agonists in a competition binding assay with A 3 membrane preparation:


agonist Known selectivity Observed IC50 (nM)
CI-IB-MECA A3 1.3
NECA Non-selective 135
CPA A1 345
CGS21680 A2A 396


Table 3. Summary of adenosine receptor antagonists in a competition binding assay with A 3 membrane preparation:


antagonist Known selectivity Observed IC50 (nM)
VUF5574 A3 2.1
DPCPX A1 759
MRS1754 A2B 1919
ZM241385 A2A 2513
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 for up to 2 years. Do not freeze and thaw.
Packaging Information
Material Size 200 units
Transport Information
Supplemental Information
Specifications

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