HTS010M
ChemiSCREEN Membrane Preparation Recombinant Rehsus Macaque CCR5 Chemokine Receptor
Rhesus Macaque CCR5 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.
Sign Into View Organizational & Contract Pricing
All Photos(1)
About This Item
UNSPSC Code:
41106514
eCl@ss:
32161000
biological source
rhesus macaque
recombinant
expressed in Chem-1 cells
manufacturer/tradename
ChemiScreen
Chemicon®
technique(s)
ligand binding assay: suitable (GTPγS)
radioligand binding assay (RLBA): suitable
NCBI accession no.
UniProt accession no.
shipped in
dry ice
Gene Information
rhesus macaque ... CCR5(735311)
General description
CCR5 is the receptor for CC chemokines MIP-1α, MIP-1β, and RANTES (Raport et al., 1996), and is preferentially expressed on Th1 lymphocytes (Loetscher et al., 1998). CCR5 is a coreceptor for macrophage-tropic HIV, and its ligands potently inhibit HIV replication in human leukocytes (Cocchi et al., 1995). In addition, HIV-infected patients with the nonfunctional CCR5Δ32 allele exhibit delayed onset of AIDS symptoms (Samson et al., 1996), and pharmacological antagonism of CCR5 inhibits HIV-1 infection (Strizki et al., 2001). Preclinical testing of small molecule antagonists of CCR5 has been hampered by low affinity of the compounds to rodent and dog CCR5, but two such compounds, maraviroc and AD101, have been shown to have potent antagonist activity at rhesus macaque CCR5, which differs from human CCR5 by 8 amino acids (Napier et al., 2005; Billick et al., 2004). One antagonist of human CCR5, SCH-C, does not block HIV entry through rhesus macaque CCR5, and one amino acid difference is responsible for the functional difference (Billick et al., 2004). Millipore′s rhesus macaque CCR5 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 rCCR5. The membrane preparations exhibit a Kd of 0.32 nM for [125I]-MIP-1α. With 0.25 nM [125I]-MIP-1α, 5 μg/well rCCR5 Membrane Prep typically yields greater than 7-fold signal-to-background ratio.
Full-length rhesus macaque CCR5 cDNA encoding CCR5
Application
Radioligand binding assay and GTPγS binding.
Biochem/physiol Actions
GPCR Class: A
Protein Target: CCR5
Target Sub-Family: Chemokine
Quality
Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of CCR5 Receptor membrane prep.
SPECIFICATIONS: 1 unit = 5 µg membrane preparation
Bmax for [125I]-MIP-1α binding: 1.9 pmol/mg protein
Kd for [125I]-MIP-1α binding: ~0.35 nM
| 10 µg/well | 5 µg/well | |
|---|---|---|
| Signal:Background | 13.6 | 12.1 |
| Specific Binding (cpm) | 5869 | 4538 |
SPECIFICATIONS: 1 unit = 5 µg membrane preparation
Bmax for [125I]-MIP-1α binding: 1.9 pmol/mg protein
Kd for [125I]-MIP-1α binding: ~0.35 nM
Specifications
Inucbation Conditions
RECOMMENDED ASSAY 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, an FC 96-well harvest plate (Millipore cat. # MAHF C1H) is coated with 0.33% polyethyleneimine for 30 min, then washed with 50mM HEPES, pH 7.4, 0.5% BSA. 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 Hepes, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.2% BSA, filtered and stored at 4°C
Radioligand: [125I]-MIP-1α (Perkin Elmer#: NEX-298 )
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, 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 7-fold signal:background with 125I labeled MIP-1α at 0.15 nM
RECOMMENDED ASSAY 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, an FC 96-well harvest plate (Millipore cat. # MAHF C1H) is coated with 0.33% polyethyleneimine for 30 min, then washed with 50mM HEPES, pH 7.4, 0.5% BSA. 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 Hepes, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.2% BSA, filtered and stored at 4°C
Radioligand: [125I]-MIP-1α (Perkin Elmer#: NEX-298 )
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, 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 7-fold signal:background with 125I labeled MIP-1α at 0.15 nM
Physical form
Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.
Packaging method: Membranes protein were adjusted to 1 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80°C
Packaging method: Membranes protein were adjusted to 1 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80°C
Storage and Stability
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
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.
Storage Class
12 - Non Combustible Liquids
wgk_germany
WGK 2
flash_point_f
Not applicable
flash_point_c
Not applicable
Certificates of Analysis (COA)
Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.
Already Own This Product?
Find documentation for the products that you have recently purchased in the Document Library.
Active Filters
Our team of scientists has experience in all areas of research including Life Science, Material Science, Chemical Synthesis, Chromatography, Analytical and many others.
Contact Technical Service
