ChemiSCREEN™ Membrane Preparation Recombinant Human P2Y₁₂ Purinergic Receptor

ChemiSCREEN™ Membrane Preparation Recombinant Human P2Y₁₂ Purinergic Receptor

• Chemicon (Millipore)
• ChemiScreen

200 units

Human P2Y12

P2Y₁₂ is a G_i-coupled 7TM receptor belonging to the P2Y family of nucleotide receptors. The principle ligand for P2Y₁₂ in vivo is ADP, although it binds with lesser affinity to ATP (von Kügelgen, 2006). P2Y₁₂ is expressed predominantly in platelets, where it mediates ADP-induced stable aggregation and secretion (Michelson, 2008). The antithrombotic drugs clopidogrel and ticlopidine disrupt P2Y₁₂-mediated platelet aggregation, and are indicated in acute coronary syndromes. However, metabolites of each drug that are generated by hepatic cytochrome P450 activity are the actual entities that bind and antagonize P2Y₁₂ directly. Novel P2Y₁₂ antagonists that bind directly to P2Y12 exhibit a faster onset of action, and are undergoing clinical trials.
Millipore's P2Y₁₂ Membrane Preparation is a crude membrane preparation made from the proprietary stable recombinant cell line that is also used for Millipore’s P2Y₁₂ Calcium Optimized Cell Line (HTS195C). This P2Y₁₂ Membrane Preparation has been optimized for low basal activity in GTPγS binding assays; thus it is an ideal HTS tool for screening of compounds for agonist and antagonist activities at P2Y₁₂. The membrane preparation exhibits EC50 of 500 nM for ADP and 26.9 nM for 2-methylthio-ADP (2-MeSADP) in a GTPγS binding assay.

Radioligand binding assay and GTPγS binding

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.

Human

1 unit = 5 μg
EC50 in GTPγS binding assay : ~ 500nM for ADP
~ 26.8nM for 2-Methylthio-ADP

Chem-1

Chemicon

Membranes are permeabilized by addition of saponin to an equal concentration by mass, then mixed with [³⁵S]-GTPγS (final concentration of 0.3 nM) in 20 mM HEPES, pH 7.4/100 mM NaCl/10 mM MgCl₂/0.5 μM GDP in a nonbinding 96-well plate. Unlabeled ligand was added to the final concentration indicated in Figure 1 (final volume 100 μL), and incubated for 30 min at 30°C. The binding reaction is transferred to an FB filter plate (Millipore MAHF B1H) previously prewetted with water. The plate is washed 3 times (1 mL per well per wash) with cold 10 mM sodium phosphate, pH 7.4, then dried and counted.

One package contains enough membranes for at least 200 assays (units), where one unit is the amount of membrane that will yield greater than 1000 cpm specific ADP-stimulated [³⁵S]-GTPγS binding.

U-2 OS

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Packaging method: Membrane protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80°C.

P2Y12

non-peptide

Store at –70°C. Product is stable for at least 6 months from the date of receipt when stored as directed. Do not freeze and thaw.

P2Y / Purinergic (metabotropic)

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A

GPCR Membrane Preparations

ChemiSCREEN™ Membrane Preparation Recombinant Human P2Y₁₂ Purinergic Receptor

NM_020400

GPCR