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Figure 1. Comparison of specific binding obtained in saturation binding with EP3 membrane preparation in harvest plate and Ready-To-Assay ...
Figure 2. Comparison of Harvest Plate and Frozen Preplated Membrane competition binding methods with EP3 membrane preparation.
ChemiSCREEN™ EP3 Prostanoid Receptor Ready-to-Assay Plates Frozen Pre-Plated Membrane Preparation (96-well)
Description:
ChemiSCREEN™ EP3 Prostanoid Receptor Ready-to-Assay Plates Frozen Pre-Plated Membrane Preparation (96-well)
Trade Name:
Chemicon (Millipore)
Qty/Pk:
1 plate
Product Overview:
Full-length human PTGER3 cDNA encoding splice variant 6 of EP3
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. Millipore currently offers MultiScreenHTS FB and FC glass fiber filter plates with deep wells that permit the binding reaction to be performed in the filter plate, thereby eliminating the need for a separate assay plate and for a transfer step. Millipore has now developed Ready-To-Assay plates: MultiScreenHTS FC plates preplated with ChemiScreenTM GPCR membrane preparations at an optimized amount (1 unit) per well. Ready-To-Assay plates free the user from pretreating filter plates and dispensing membranes onto the plates, and provide signal:background values equal to or better than those obtained with a conventional harvest plate method. Millipore offers plates with either a single receptor per plate or with an array of related receptors on one plate.
Prostanoids bind to a family of 8 GPCRs to exert their biological effects (Narumiya and FitzGerald, 2001). 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. Further diversity is generated by alternative splicing; the human gene for EP3 generates 9 alternatively spliced mRNAs encoding 8 isoforms of EP3 (Kotani et al., 1997). These isoforms of EP3 vary in sequence at their C-termini, and differ in their ability to couple to Gs, Gq or Gi (Kotani et al., 1995). EP3 is required for fever induced by pyrogens, a response long attributed to prostaglandins by the antipyretic action of aspirin and other COX inhibitors (Ushikubi et al., 1998). Chemicon's EP3 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 EP3 interactions and its ligands.
The Recombinant Human EP3 Frozen Preplated Membrane Preparations (96-well) exhibit a signal-to-background ratio of greater than 40-fold with 1.5 nM [3H]-PGE2, which is comparable to that observed with a traditional harvest plate method.
Prostanoids bind to a family of 8 GPCRs to exert their biological effects (Narumiya and FitzGerald, 2001). 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. Further diversity is generated by alternative splicing; the human gene for EP3 generates 9 alternatively spliced mRNAs encoding 8 isoforms of EP3 (Kotani et al., 1997). These isoforms of EP3 vary in sequence at their C-termini, and differ in their ability to couple to Gs, Gq or Gi (Kotani et al., 1995). EP3 is required for fever induced by pyrogens, a response long attributed to prostaglandins by the antipyretic action of aspirin and other COX inhibitors (Ushikubi et al., 1998). Chemicon's EP3 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 EP3 interactions and its ligands.
The Recombinant Human EP3 Frozen Preplated Membrane Preparations (96-well) exhibit a signal-to-background ratio of greater than 40-fold with 1.5 nM [3H]-PGE2, which is comparable to that 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:
The protein encoded by this gene is a member of the G-protein coupled receptor family. This protein is one of four receptors identified for prostaglandin E2 (PGE2). This receptor may have many biological functions, which involve digestion, nervous system, kidney reabsorption, and uterine contraction activities. Studies of the mouse counterpart suggest that this receptor may also mediate adrenocorticotropic hormone response as well as fever generation in response to exogenous and endogenous stimuli. Multiple alternatively spliced transcript variants encoding eight distinct isoforms have been reported.
UniProt Summary:
FUNCTION: SwissProt: P43115 # Receptor for prostaglandin E2 (PGE2); the EP3 receptor may be involved in inhibition of gastric acid secretion, modulation of neurotransmitter release in central and peripheral neurons, inhibition of sodium and water reabsorption in kidney tubulus and contraction in uterine smooth muscle. The activity of this receptor can couple to both the inhibition of adenylate cyclase mediated by G-I proteins, and to an elevation of intracellular calcium. The various isoforms have identical ligand binding properties but can interact with different second messenger systems (By similarity).
SIZE: 390 amino acids; 43310 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Expressed at least in small intestine, heart and pancreas.
SIMILARITY: SwissProt: P43115 ## Belongs to the G-protein coupled receptor 1 family.
SIZE: 390 amino acids; 43310 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Expressed at least in small intestine, heart and pancreas.
SIMILARITY: SwissProt: P43115 ## Belongs to the G-protein coupled receptor 1 family.
Species:
Human
Quality Assurance:
Table 1. Signal:background, Kd and Bmax values for EP3 prostanoid receptor obtained in competition and 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. A signal:background ratio of greater than 40-fold was obtained.
Harvest Plate method: EP3 membrane preparation (Chemicon cat. #HTS092M) was thawed rapidly and chilled on ice. A binding reaction consisting of unlabeled PGE2 at the concentration indicated, 1.5 nM [3H]-PGE2 and 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 on top read mode.
| Ready-To-Assay Plate method | Harvest plate method | |
| Kd (nM) from saturation binding | 4.65 | 2.6 |
| Bmax (pmol/mg) from saturation binding | 33.18 | 21.7 |
| Signal:background from competition binding | 132.5 | 51.8 |
| Z' | 0.73 | 0.75 |
Frozen Preplated Membrane Method: Performed as described in "RECOMMENDED ASSAY PROTOCOL FOR COMPETITION BINDING" below. A signal:background ratio of greater than 40-fold was obtained.
Harvest Plate method: EP3 membrane preparation (Chemicon cat. #HTS092M) was thawed rapidly and chilled on ice. A binding reaction consisting of unlabeled PGE2 at the concentration indicated, 1.5 nM [3H]-PGE2 and 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 on top read mode.
Cell Type:
Chem-1
Brand Family:
Chemicon
Incubation Conditions:
RECOMMENDED ASSAY PROTOCOL FOR COMPETITION BINDING:
1. Thaw the plate in a 37°C incubator with the lid in place.
2. Add to the thawed membranes in the plate: radiolabeled ligand ([3H] PGE2, Perkin Elmer #NET-428, diluted in binding buffer for a final concentration of 1.5 nM), 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, 300 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 Tris, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 0.2% BSA, filtered and stored at 4°C
Wash Buffer: 50 mM Tris, pH 7.4 filtered and stored at 4°C.
Plate is tested to ensure that Z'≥0.5 and signal:background ratio≥40-fold with [3H] PGE2.
1. Thaw the plate in a 37°C incubator with the lid in place.
2. Add to the thawed membranes in the plate: radiolabeled ligand ([3H] PGE2, Perkin Elmer #NET-428, diluted in binding buffer for a final concentration of 1.5 nM), 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, 300 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 Tris, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 0.2% BSA, filtered and stored at 4°C
Wash Buffer: 50 mM Tris, pH 7.4 filtered and stored at 4°C.
Plate is tested to ensure that Z'≥0.5 and signal:background ratio≥40-fold with [3H] PGE2.
Host Cells:
Chem-1
Presentation:
EP3 membranes 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:
EP3
UniProt Number:
Ligand Type:
non-peptide
Storage Conditions:
Maintain frozen at –70°C up to expiration date indicated on the label. Do not freeze and thaw.
Target Sub-Family:
Prostanoid
Promotional Text:
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GPCR Class:
A
Gene Symbol:
- PTGER3
- EP3-III
- EP3-I
- PGE2-R
- MGC141829
- EP3-II
- EP3
- EP3e
- MGC27302
- MGC141828
- EP3-IV
Protein or Enzyme Type:
GPCR Membrane Preparations
Product Name:
ChemiSCREEN™ EP3 Prostanoid Receptor Ready-to-Assay Plates Frozen Pre-Plated Membrane Preparation (96-well)
Entrez Gene Number:
Cell Membrane Type:
GPCR