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Figure 1. Comparison of harvest plate and Ready-To-Assay plate methods in saturation binding with Prostanoid Receptors.
ChemiSCREEN™ Prostanoid Receptor Array Ready-to-Assay Plates Frozen Pre-Plated Membrane Preparation (96-well)
Description:
ChemiSCREEN™ Prostanoid Receptor Array Ready-to-Assay Plates Frozen Pre-Plated Membrane Preparation (96-well)
Trade Name:
- ChemiScreen
- Chemicon (Millipore)
Qty/Pk:
1 plate
Background Information:
Traditional GPCR radioligand binding assays performed by filtration require assembly of the binding reaction in an assay plate, followed by transfer of the reaction to a PEI-coated glass fiber filter plate for filtration. Millipore currently offers MultiScreenHTS FB and FC plates with a deep well that permits the binding reaction to be performed in the filter plate, thereby eliminating the need for separate filtration and assay plates. Millipore has now developed Ready-To-Assay Plates, which contain ChemiScreen GPCR membrane preparations pre-frozen on a MultiScreenHTS FC plate at an optimized amount of 1 unit per well.
Prostanoids are a series of arachidonic acid metabolites produced by the action of cyclooxygenase and further modified by isomerases and synthases. Cells rapidly secrete prostanoids after synthesis, whereupon the prostanoids bind to a family of 8 GPCRs to exert their biological effects (Narumiya and FitzGerald, 2001; Hata and Breyer, 2004). Millipore's Prostanoid Receptor Array contains membrane preparations for two prostaglandin E2 receptors (EP3 and EP4), two prostaglandin D2 receptors (DP and CRTH2), and the thromboxane A2 receptor (TP). The prostaglandin PGE2 causes pain, vasodilation, immunosuppression of T cells, bone resorption and promotion of carcinogenesis. Four related GPCRs, EP1, EP2, EP3 and EP4, each bind to PGE2, but the different G protein coupling status of each receptor leads to distinct biological effects. The prostaglandin PGD2 is produced by mast cells upon activation by allergens, and is present at high levels in allergic diseases. PGD2 binds to two receptors, DP and CRTH2. Thromboxane A2 receptor (TP) is widely distributed among different organ systems and have been localized on both cell membranes and intracellular structures. TP receptors belong to G protein-coupled receptor family (Hirata et al., 1991). Activation of TP receptors induces platelet aggregation and vascular and respiratory smooth muscle constriction and cell growth. Chemicon's Prostanoid Receptor 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 prostanoid receptors.
The Recombinant Human Prostanoid Receptor Array (96-well) exhibits comparable signal-to-background ratios, and Kd and Bmax values to those observed with a traditional harvest plate method.
Prostanoids are a series of arachidonic acid metabolites produced by the action of cyclooxygenase and further modified by isomerases and synthases. Cells rapidly secrete prostanoids after synthesis, whereupon the prostanoids bind to a family of 8 GPCRs to exert their biological effects (Narumiya and FitzGerald, 2001; Hata and Breyer, 2004). Millipore's Prostanoid Receptor Array contains membrane preparations for two prostaglandin E2 receptors (EP3 and EP4), two prostaglandin D2 receptors (DP and CRTH2), and the thromboxane A2 receptor (TP). The prostaglandin PGE2 causes pain, vasodilation, immunosuppression of T cells, bone resorption and promotion of carcinogenesis. Four related GPCRs, EP1, EP2, EP3 and EP4, each bind to PGE2, but the different G protein coupling status of each receptor leads to distinct biological effects. The prostaglandin PGD2 is produced by mast cells upon activation by allergens, and is present at high levels in allergic diseases. PGD2 binds to two receptors, DP and CRTH2. Thromboxane A2 receptor (TP) is widely distributed among different organ systems and have been localized on both cell membranes and intracellular structures. TP receptors belong to G protein-coupled receptor family (Hirata et al., 1991). Activation of TP receptors induces platelet aggregation and vascular and respiratory smooth muscle constriction and cell growth. Chemicon's Prostanoid Receptor 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 prostanoid receptors.
The Recombinant Human Prostanoid Receptor Array (96-well) exhibits comparable signal-to-background ratios, and Kd and Bmax values to those observed with a traditional harvest plate method.
Application Notes:
Radioligand binding assay
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.
Entrez Gene Summary:
G protein-coupled receptors (GPCRs), such as GPR44, are integral membrane proteins containing 7 putative transmembrane domains (TMs). These proteins mediate signals to the interior of the cell via activation of heterotrimeric G proteins that in turn activate various effector proteins, ultimately resulting in a physiologic response.[supplied by OMIM]
UniProt Summary:
FUNCTION: SwissProt: Q9Y5Y4 # Orphan receptor.
SIZE: 395 amino acids; 43240 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
SIMILARITY: SwissProt: Q9Y5Y4 ## Belongs to the G-protein coupled receptor 1 family.
SIZE: 395 amino acids; 43240 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
SIMILARITY: SwissProt: Q9Y5Y4 ## Belongs to the G-protein coupled receptor 1 family.
Species:
Human
Quality Assurance:
Table 1. Kd and Bmax values for prostanoid receptors obtained in saturation binding with harvest plate and Ready-To-Assay plate methods
Frozen Preplated Membrane Method: Performed as described in "RECOMMENDED ASSAY PROTOCOL FOR COMPETITION BINDING" below.
Harvest Plate method: Membrane preparations (Chemicon cat. #) were thawed rapidly and chilled on ice. Binding reactions consisting of unlabeled and labeled ligands at the concentrations indicated, 10 ug/well membrane preparation in binding buffer was assembled in an assay plate (Corning). The reaction was incubated for 2 h at room temperature, during which time a MultiScreen Harvest Plate (Millipore cat. # MAHF C1H) was incubated for 15 min with 0.3% PEI and washed with 50mM HEPES, pH 7.4, 0.5% BSA. Binding reaction was transferred to the filter plate, and washed 3 times (1 mL per well per wash) with Wash Buffer. The plate was dried and counted in top read mode.
| Kd (nM) with Ready-To-Assay Plate | Kd (nM) with harvest plate method | Bmax with Ready-To-Assay plate (pmol/mg) | Bmax with harvest plate method (pmol/mg) | |
| DP | 6.6 | 5.9 | 15.2 | 10.3 |
| CRTH2 | 10.2 | 9.1 | 25.1 | 26.3 |
| TP | 12.7 | 25.9 | 55.4 | 77.1 |
| EP3 | 2.5 | 3.2 | 28.8 | 33.5 |
| EP4 | 4.2 | 12.6 | 6.5 | 10.1 |
Harvest Plate method: Membrane preparations (Chemicon cat. #) were thawed rapidly and chilled on ice. Binding reactions consisting of unlabeled and labeled ligands at the concentrations indicated, 10 ug/well membrane preparation in binding buffer was assembled in an assay plate (Corning). The reaction was incubated for 2 h at room temperature, during which time a MultiScreen Harvest Plate (Millipore cat. # MAHF C1H) was incubated for 15 min with 0.3% PEI and washed with 50mM HEPES, pH 7.4, 0.5% BSA. Binding reaction was transferred to the filter plate, and washed 3 times (1 mL per well per wash) with Wash Buffer. The plate was dried and counted in top read mode.
Cell Type:
Chem-1
Brand Family:
Chemicon
Incubation Conditions:
RECOMMENDED ASSAY PROTOCOL FOR COMPETITION BINDING:
1. Thaw the plate at room temperature with the lid in place.
2. Add to the thawed membranes in the plate: radiolabeled ligand (see table 1 below for specific ligand and concentration), test compound and binding buffer for a final volume of 100 µL (the volume of preplated GPCR membrane is 25 uL).
3. Cover the plate with the lid and incubate for 1-2 h at room temperature.
4. Stop reaction by addition of 100 µL/well binding buffer.
5. Filter with MultiScreenHTS Vacuum Manifold.
6. Wash 3x with Wash Buffer, 200 µL/well/wash.
7. Remove the underdrain from the bottom of the plate.
8. Dry the filter plate.
9. Seal the bottom with clear tape
10. Add 50 µL/well scintillation cocktail.
11. Seal the top of plate with clear tape, and count in Microbeta scintillation counter on coincidence mode
Binding Buffer: 50 mM Hepes pH 7.4, 5 mM MgCl2, 1 mM CaCl2, , 0.2% BSA, filtered and stored at 4°C
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA filtered and stored at 4°C.
Table 3. Radioligands for binding assay with Prostanoid Receptor Array
1. Thaw the plate at room temperature with the lid in place.
2. Add to the thawed membranes in the plate: radiolabeled ligand (see table 1 below for specific ligand and concentration), test compound and binding buffer for a final volume of 100 µL (the volume of preplated GPCR membrane is 25 uL).
3. Cover the plate with the lid and incubate for 1-2 h at room temperature.
4. Stop reaction by addition of 100 µL/well binding buffer.
5. Filter with MultiScreenHTS Vacuum Manifold.
6. Wash 3x with Wash Buffer, 200 µL/well/wash.
7. Remove the underdrain from the bottom of the plate.
8. Dry the filter plate.
9. Seal the bottom with clear tape
10. Add 50 µL/well scintillation cocktail.
11. Seal the top of plate with clear tape, and count in Microbeta scintillation counter on coincidence mode
Binding Buffer: 50 mM Hepes pH 7.4, 5 mM MgCl2, 1 mM CaCl2, , 0.2% BSA, filtered and stored at 4°C
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA filtered and stored at 4°C.
Table 3. Radioligands for binding assay with Prostanoid Receptor Array
| radioligand | Recommended concentration for competition | Vendor/part number | |
| DP | [3H]-Prostaglandin D2 | 5 nM | Perkin Elmer NET616; Amersham TRK734 |
| CRTH2 | [3H]-Prostaglandin D2 | 10 nM | Perkin Elmer NET616; Amersham TRK734 |
| TP | [3H]- SQ 29548 | 10 nM | Perkin Elmer NET936 |
| EP3 | [3H]-Prostaglandin E2 | 1.5 nM | Perkin Elmer NET428: Amersham TRK431 |
| EP4 | [3H]-Prostaglandin E2 | 3 nM | Perkin Elmer NET428: Amersham TRK431 |
Host Cells:
Chem-1
Presentation:
GPCR membranes preparations are supplied at 10 µg in 25 µL (1 unit) per well, in each well of the plate. The membranes are formulated in 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Protein Target:
DP2, DP, EP3, EP4, TP
UniProt Number:
Ligand Type:
non-peptide
Storage Conditions:
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
Target Sub-Family:
Prostanoid
Promotional Text:
For special offers Click here http://www.millipore.com/drugdiscovery/dd3/gpcrtargetsolutions
GPCR Class:
A
Packaging:
One 96-well plate containing EP3, EP4, DP, CRTH2 and TP membrane preparations
Gene Symbol:
- PTGDR
- DP
- MGC49004
- AS1
- ASRT1
Protein or Enzyme Type:
GPCR Membrane Preparations
Product Name:
ChemiSCREEN™ Prostanoid Receptor Array Ready-to-Assay Plates Frozen Pre-Plated Membrane Preparation (96-well)
Entrez Gene Number:
Cell Membrane Type:
GPCR