Membrane Preparations
Purified GPCR and Ion Channel Membrane Preps for Binding and Functional Analysis
Membrane preps are useful for studying the role of membrane proteins in diseases and their response to therapeutics. Eurofins DiscoverX® GPCR and ion channel membrane preps are derived from stable cell lines expressing optimal levels of the protein to be assayed. They are designed for determining membrane protein binding affinities through saturation and competitive radioligand binding analysis, and can be used for rank ordering and screening of small molecule or antibody therapeutics. For GPCRs, these membrane preps can also be used to perform GTPγS functional studies to evaluate GPCR activity in response to the addition of a ligand or therapeutic.
Utilize Eurofins DiscoverX’s purified membrane preps for highly sensitive and reproducible results you can rely on.
Product Highlights
- Robust Performance – Large signal-to-background ratios with specific and high ligand binding capacity
- Reproducible – Produced from stable clonal cell lines for superior protein expression and lot-to-lot consistency
- Versatile Use – Applicable for saturation and competitive radioligand binding, therapeutic small molecule and antibody screening and ranking, and GTPγS functional studies
- ChemiSCREEN™ GPCRs Membrane Preps – Purified cell membrane preps expressing high levels of specific target GPCR proteins are applicable for determining GPCR activity via GTPγS assay and binding affinities via saturation and competitive binding assays.
- PrecisION® Ion Channel (hERG) Membrane Prep – Purified cell membrane prep expressing high levels of human ERG voltage-gated ion channel for the determination of binding affinities via saturation and competitive binding assays.
GTPyS Assay Principle
GTPγS assays are assays intended to determine the amount of GPCR activation in response to ligand binding. Upon addition of both ligand and GTPγS, the GPCR engages with its G-protein complex and results in the release of GDP. GTPγS is a nonhydrolyzable GTP analogue that will have a longer persistence time at the G-protein. Since the assay is provided with only GTPγS and not normal GTP, this GTPγS is incorporated into the GPCR G-protein complex. The inclusion of a radioactive 35S atom in GTPγS signifies that radioactivity will be produced in the assay directly proportional to the number of ligand-activated GPCRs.
Competitive Ligand Binding Assay Principle
Competitive ligand binding assays use a constant concentration of radiolabeled ligand (commonly conjugated to 125I) and a varied concentration of unlabeled ligand. When unlabeled ligand is low, most of the membrane proteins will be bound by radiolabeled ligand, and the detected signal will be high. As the amount of unlabeled ligand increases, more and more of the membrane proteins will be bound by unlabeled ligand, and the detected signal will decrease. The binding affinity can be calculated from the dose-response curve generated with varying ligand concentrations tested during the assay.
Saturation and Competitive Binding Analysis of Cannabinoid Receptor 1
Ligand binding analysis of cannabinoid receptor 1 (CB1) (Cat. No. HTS019M). A. Saturation binding: 7.5 μg/well of CB1 membrane was incubated with increasing amounts of [3H]-CP 55,940 in the absence (total binding, TB) or presence (nonspecific binding, NSB) of 5000-fold excess unlabeled WIN55212 (a CB1 agonist). Specific binding (SB) was determined by subtracting NSB from TB. B. Competition binding: 7.5 μg/well of CB1 membrane or wild type (WT) (Cat. No. HTS000MC1) Chem-1 (cell type) membrane was incubated with 2nM [3H]-CP 55,940 and increasing concentrations of unlabeled CP 55,940, then subjected to filtration binding. Results show a greater than 1.5-fold signal:background ratio was obtained. (dpm = disintegrations per minute)
GPCR Activity Analysis of Cannabinoid Receptor 2
GPCR activity analysis of cannabinoid receptor 2 (CB2) (Cat. No. HTS020M): 5 μg/well of CB2 or WT Chem-1 membrane prep (Cat. No. HTS000MC1) was incubated with 0.3 nM [35S]-GTPyS, 10 μM GDP, and increasing amounts of unlabeled CP 55,940 (ligand). Bound radioactivity was then determined by filtration and scintillation counting. Results show an increasing % of bound GTPγS, indicating increased G-protein activation with increasing amounts of added CP 55,940.
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