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Figure 1. Comparison of specific binding obtained in saturation binding with TP membrane preparation in harvest plate and Ready-To-Assay plate method...
Figure 2. Comparison of Harvest Plate and Frozen Preplated Membrane competition binding methods with TP membrane preparation.
ChemiSCREEN™ TP Prostanoid Receptor Ready-to-Assay Plates Frozen Pre-Plated Membrane Preparation (96-well)
Description:
ChemiSCREEN™ TP 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 TBXA2R cDNA encoding the TP short form (TPα)
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.
TP receptors (Thromboxane A2 receptors) are 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, vascular and respiratory smooth muscle constriction, and enhances mitogenic responses of vascular smooth muscle cells that are stimulated by growth factors (Ali et al, 1993; Hanasaki et al., 1990). The human TP receptor is encoded by a single gene that is alternatively spliced at the carboxyl terminus, resulting in two isoforms, TPα (343 residues) and TPβ (407 residues). Both isoforms couple to Gq pathway, but couple oppositely to adenylate cyclase. Millipore's TP 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 TP interactions and its ligands.
The Recombinant Human TP Frozen Preplated Membrane Preparations (96-well) exhibit a signal-to-background ratio of greater than 15-fold with 10 nM [3H]-SQ29548, which is comparable to that observed with a traditional harvest plate method.
TP receptors (Thromboxane A2 receptors) are 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, vascular and respiratory smooth muscle constriction, and enhances mitogenic responses of vascular smooth muscle cells that are stimulated by growth factors (Ali et al, 1993; Hanasaki et al., 1990). The human TP receptor is encoded by a single gene that is alternatively spliced at the carboxyl terminus, resulting in two isoforms, TPα (343 residues) and TPβ (407 residues). Both isoforms couple to Gq pathway, but couple oppositely to adenylate cyclase. Millipore's TP 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 TP interactions and its ligands.
The Recombinant Human TP Frozen Preplated Membrane Preparations (96-well) exhibit a signal-to-background ratio of greater than 15-fold with 10 nM [3H]-SQ29548, 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.
UniProt Summary:
FUNCTION: SwissProt: P21731 # Receptor for thromboxane A2 (TXA2), a potent stimulator of platelet aggregation. The activity of this receptor is mediated by a G-protein that activate a phosphatidylinositol-calcium second messenger system. In the kidney, the binding of TXA2 to glomerular TP receptors causes intense vasoconstriction.
SIZE: 369 amino acids; 40092 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
DISEASE:SwissProt: P21731 # Defects in TBXA2R are the cause of a dominantly inherited bleeding disorder [MIM:188070].
SIMILARITY: SwissProt: P21731 ## Belongs to the G-protein coupled receptor 1 family.
SIZE: 369 amino acids; 40092 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
DISEASE:SwissProt: P21731 # Defects in TBXA2R are the cause of a dominantly inherited bleeding disorder [MIM:188070].
SIMILARITY: SwissProt: P21731 ## Belongs to the G-protein coupled receptor 1 family.
Species:
Human
Quality Assurance:
Table 1. Signal:background, Kd and Bmax values for EP4 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 15-fold was obtained.
Harvest Plate method: TP membrane preparation (Chemicon cat. #HTS081M) was thawed rapidly and chilled on ice. A binding reaction consisting of unlabeled ICI 192,605 at the concentration indicated, 10 nM [3H]-SQ29548 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 | 12.02 | 13.37 |
| Bmax (pmol/mg) from saturation binding | 18.3 | 10.5 |
| Signal:background from competition binding | 24.8 | 11 |
| Z' | 0.73 | 0.76 |
Harvest Plate method: TP membrane preparation (Chemicon cat. #HTS081M) was thawed rapidly and chilled on ice. A binding reaction consisting of unlabeled ICI 192,605 at the concentration indicated, 10 nM [3H]-SQ29548 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≥15-fold with [3H]-SQ29548.
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≥15-fold with [3H]-SQ29548.
Host Cells:
Chem-1
Presentation:
TP 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:
TP / TXA2 / PGH2
Format:
Membranes
UniProt Number:
Ligand Type:
non-peptide
Target Sub-Family:
Prostanoid
Storage Conditions:
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
Promotional Text:
For special offers Click here http://www.millipore.com/drugdiscovery/dd3/gpcrtargetsolutions
GPCR Class:
A
Gene Symbol:
- TBXA2R
- TXA2-R
Protein or Enzyme Type:
GPCR Membrane Preparations
Product Name:
ChemiSCREEN™ TP Prostanoid Receptor Ready-to-Assay Plates Frozen Pre-Plated Membrane Preparation (96-well)
Entrez Gene Number:
Cell Membrane Type:
GPCR