ChemiSCREEN™ CCR10 Membrane Preparation

ChemiSCREEN™ CCR10 Membrane Preparation

ChemiScreen

Full length human CCR10 with a proprietary mutation in the cytoplasmic domain to enhance cell surface expression.

CCR10, previously known as GPR2, is a GPCR that is expressed in dermal endothelial cells, fibroblasts, melanocytes, and circulating T cells and PBMCs (Homey et al., 2000). Two chemokines, CTACK/CCL27 and MEC/CCL28, bind to CCR10 and stimulate migration and calcium flux (Pan et al., 2000; Wang et al., 2000). Expression of CTACK by keratinocytes, and CCR10 by lymphocytes from skin of patients with psoriasis and dermatitis, implicates this receptor ligand pair in skin inflammation. In addition, disruption of CCL27-CCR10 interaction reduces skin inflammation induced by allergen (Homey et al., 2002). CCR10 expressed on IgA antibody-secreting cells also mediates their migration to CCL28 expressed in the lactating mammary gland, and subsequent IgA accumulation in milk (Wilson and Butcher, 2004). Millipore's CCR10 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 CCR10 interactions with its ligands. The membrane preparations exhibit an EC50 of 47 nM for CTACK/CCL27 in a GTPγS binding assay.

Radioligand binding assay and GTPgammaS binding.

RECOMMENDED ASSAY CONDITIONS:

Binding buffer: 50mM HEPES, 5mM MgCl2, 1mM CaCl2, 0.2% BSA, pH to 7.4 and filter.

Radioligand: [125I] CTACK/CCL27 (PerkinElmer#: NEX391)

Human

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.

1.0 mg/mL

Chem-1

• CCR9
• CC-CKR-9
• D6
• GPR-9-6
• CDw199
• CCBP2
• GPR28
• CCR10
• CCR-9
• CMKBR9

Membranes

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Packaging method: Membranes protein were adjusted to 1 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80°C.

EC50 for CTACK/CCL27: ~47 nM in a GTPγS binding assay

Membrane Preparations

ASSAY CONDITIONS: Membranes are 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 CTACK/CCL27 is 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 vial 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 CTACK/CCL27-stimulated [35S]-GTPγS binding. The CCR10 membrane preparation is expected to be functional in a radioligand binding assay; however, the end user will need to determine the optimal radiolabeled ligand for use with this product.

Human

Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.