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Figure 1. Saturation binding for A3. 5 µg/well A3 Membrane Preparation was incubated with increasing amount of 125I-labeled AB-MECA in the absence ( ...
Figure 3. Rank order of binding of adenosine receptor agonists and antagonists to A3 Membrane Preparation. A3 Membrane Prepara ...
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ChemiScreen™ A3 Membrane Preparation
Description:
ChemiScreen™ A3 Membrane Preparation
Trade Name:
ChemiScreen
Product 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.
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.
Application Notes:
Radioligand binding assay and GTPγS binding.
Species Reactivity:
Human
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.
Concentration:
1 mL, 1.0 mg/mL
Host Cells:
Chem-3, a suspension mammalian cell line without any endogenous A3 expression.
Cell Type:
Chem-3
Gene Symbol:
- ADORA3
- AD026
- bA552M11.5
- ORFNames=UNQ1931/PRO4406
- RP11-552M11.7
- A3AR
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.
Quality Assurance:
Table 1. Signal:background and specific binding values obtained in a competition binding assay with A3 membrane preparation:
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 A3 membrane preparation:
Table 3. Summary of adenosine receptor antagonists in a competition binding assay with A3 membrane preparation:
| 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 A3 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 A3 membrane preparation:
| antagonist | Known selectivity | Observed IC50 (nM) |
|---|---|---|
| VUF5574 | A3 | 2.1 |
| DPCPX | A1 | 759 |
| MRS1754 | A2B | 1919 |
| ZM241385 | A2A | 2513 |
Protein or Enzyme Type:
Membrane Preparations
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
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
Species:
Human
Storage Conditions:
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.