ChemiSCREEN Membrane Preparation Recombinant Human MrgX1 Receptor

Human MrgX1

The Mas-related gene family (Mrg), also known as SNSR (sensory neuron-specific G protein-coupled receptors), are expressed mainly in the dorsal root ganglia and appear to be involved in nociception and other sensory processes. MrgX1/SNSR4 is activated by the neuropeptide BAM22, and it mediates neuronal synaptic transmission and ion channel activity (Lembo et al., 2002; Burstein et al., 2006, Chen and Ikeda, 2004). Millipore′s MrgX1 Membrane Preparation for GTPγS Assay is a crude membrane preparation made from the proprietary stable recombinant cell line that is also used for Millipore′s MrgX1 Calcium Optimized Cell Line (HTS122C). This MrgX1 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. The membrane preparations exhibits EC50 of 93.7 nM for BAM(8-22) in a GTPγS binding assay.

Radioligand binding assay and GTPγS binding

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.

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.

1 unit = 5 μg
EC50 in GTPγS binding assay by BAM (8-22): ~ 93.7 nM

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 BAM (8-22) 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 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 BAM(8-22)-stimulated [³⁵S]-GTPγS binding.

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