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Figure 1. Comparison of specific binding obtained in saturation binding with α1A membrane preparation in harvest plate and Ready-To-Assay plate metho...
Figure 2. Comparison of Harvest Plate and Frozen Preplated Membrane competition binding methods with α1A membrane preparation.
ChemiSCREEN™ α1A Adrenergic Receptor Ready-to-Assay™ Plates Frozen Pre-Plated Membrane Preparation (96-well)
Description:
ChemiSCREEN™ α1A Adrenergic Receptor Ready-to-Assay™ Plates Frozen Pre-Plated Membrane Preparation (96-well)
Trade Name:
Chemicon (Millipore)
Qty/Pk:
1 plate
Product Overview:
Human ADR1A encoding α1A
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 ChemiScreen™ 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.
The endogenous catecholamines epinephrine and norepinephrine have profound effects on smooth muscle activity, cardiac function, carbohydrate and fat metabolism, hormone secretion, neurotransmitter release, and central nervous system actions. These activities are mediated by GPCRs belonging to two subfamilies, the α- and β-adrenoceptors (Bylund et al., 1994). The three members of the α1 subclass of adrenoceptors, α1A, α1B and α1D, couple to Gq, and promote contraction of vascular and urinary tract smooth muscle, relaxation of intestinal smooth muscle, increased contractile force in the heart, and glycogenolysis and gluconeogenesis in the liver. The different subtypes have overlapping distributions and variably contribute to these effects depending on species and tissue; the α1A subtype plays a prominent role in urogenital smooth muscle contraction and renal artery contraction (Hrometz et al., 1999; Ruffolo and Hieble, 1999). Activation of α1 adrenoceptors also influences cell proliferation; α1A inhibits cell growth by arresting progression at the G1/S transition (Shibata et al., 2003). Millipore's α1A adrenoceptor 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 α1A adrenoceptor interactions. The membrane preparations exhibit a Kd of 2.7 nM for [3H] Prazosin. With 5 μg/well α1A Adrenoceptor Membrane Prep and 1 nM [3H] Prazosin, a greater than 7-fold signal-to-background ratio was obtained.
The Recombinant Human α1A Frozen Preplated Membrane Preparations (96-well) exhibit a signal-to-background ratio of greater than 7-fold with 0.75nM [3H] Prazosin, which is comparable to that observed with a traditional harvest plate method.
The endogenous catecholamines epinephrine and norepinephrine have profound effects on smooth muscle activity, cardiac function, carbohydrate and fat metabolism, hormone secretion, neurotransmitter release, and central nervous system actions. These activities are mediated by GPCRs belonging to two subfamilies, the α- and β-adrenoceptors (Bylund et al., 1994). The three members of the α1 subclass of adrenoceptors, α1A, α1B and α1D, couple to Gq, and promote contraction of vascular and urinary tract smooth muscle, relaxation of intestinal smooth muscle, increased contractile force in the heart, and glycogenolysis and gluconeogenesis in the liver. The different subtypes have overlapping distributions and variably contribute to these effects depending on species and tissue; the α1A subtype plays a prominent role in urogenital smooth muscle contraction and renal artery contraction (Hrometz et al., 1999; Ruffolo and Hieble, 1999). Activation of α1 adrenoceptors also influences cell proliferation; α1A inhibits cell growth by arresting progression at the G1/S transition (Shibata et al., 2003). Millipore's α1A adrenoceptor 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 α1A adrenoceptor interactions. The membrane preparations exhibit a Kd of 2.7 nM for [3H] Prazosin. With 5 μg/well α1A Adrenoceptor Membrane Prep and 1 nM [3H] Prazosin, a greater than 7-fold signal-to-background ratio was obtained.
The Recombinant Human α1A Frozen Preplated Membrane Preparations (96-well) exhibit a signal-to-background ratio of greater than 7-fold with 0.75nM [3H] Prazosin, 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:
Alpha-1-adrenergic receptors (alpha-1-ARs) are members of the G protein-coupled receptor superfamily. They activate mitogenic responses and regulate growth and proliferation of many cells. There are 3 alpha-1-AR subtypes: alpha-1A, -1B and -1D, all of which signal through the Gq/11 family of G-proteins and different subtypes show different patterns of activation. This gene encodes alpha-1A-adrenergic receptor. Alternative splicing of this gene generates four transcript variants, which encode four different isoforms with distinct C-termini but having similar ligand binding properties.
UniProt Summary:
FUNCTION: SwissProt: P35348 # This alpha-adrenergic receptor mediates its action by association with G proteins that activate a phosphatidylinositol- calcium second messenger system. Its effect is mediated by G(q) and G(11) proteins.
SIZE: 466 amino acids; 51487 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Heart, brain, liver and prostate, but not in kidney, lung, adrenal, aorta and pituitary. Isoform 4 is the most abundant isoform expressed in the prostate with high levels also detected in liver and heart.
PTM: Carboxyl-terminal Ser or Thr residues may be phosphorylated (By similarity).
SIMILARITY: SwissProt: P35348 ## Belongs to the G-protein coupled receptor 1 family.
SIZE: 466 amino acids; 51487 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Heart, brain, liver and prostate, but not in kidney, lung, adrenal, aorta and pituitary. Isoform 4 is the most abundant isoform expressed in the prostate with high levels also detected in liver and heart.
PTM: Carboxyl-terminal Ser or Thr residues may be phosphorylated (By similarity).
SIMILARITY: SwissProt: P35348 ## Belongs to the G-protein coupled receptor 1 family.
Species:
Human
Quality Assurance:
Table 1. Signal:background, Kd and Bmax values for α1A adrenoceptor 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 4-fold was obtained.
Harvest Plate method: α1A membrane preparation (Chemicon cat. #HTS087M) was thawed rapidly and chilled on ice. A binding reaction consisting of unlabeled WB4101 at the concentration indicated, 1nM [3H] Prazosin and 5 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 | 2.7 | 1.4 |
| Bmax (pmol/mg) from saturation binding | 9.0 | 15.1 |
| Signal:background from competition binding | 33.9 | 8.1 |
| Z' | 0.54 | 0.53 |
Frozen Preplated Membrane Method: Performed as described in “RECOMMENDED ASSAY PROTOCOL FOR COMPETITION BINDING” below. A signal:background ratio of greater than 4-fold was obtained.
Harvest Plate method: α1A membrane preparation (Chemicon cat. #HTS087M) was thawed rapidly and chilled on ice. A binding reaction consisting of unlabeled WB4101 at the concentration indicated, 1nM [3H] Prazosin and 5 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 37ºC incubator with the lid in place.
2.Add to the thawed membranes in the plate: radiolabeled ligand ([3H] Prazosin, Perkin Elmer #NET-823, diluted in binding buffer for a final concentration of 1 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 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.
Plate is tested to ensure that Z’ ≥0.5 and signal:background ratio ≥7-fold with [3H] Prazosin.
Host Cells:
Chem-1
Presentation:
α1A membranes are supplied at 5 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:
alpha1A
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:
Adrenergic
Promotional Text:
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GPCR Class:
A
Gene Symbol:
- ADRA1A
- ADRA1C
- ADRA1L1
- ALPHA1AAR
- ADRA1D
Protein or Enzyme Type:
GPCR Membrane Preparations
Product Name:
ChemiSCREEN™ α1A Adrenergic Receptor Ready-to-Assay™ Plates Frozen Pre-Plated Membrane Preparation (96-well)
Entrez Gene Number:
Cell Membrane Type:
GPCR