Scintillation Proximity Assay

Kahl, Steven D.; Felder, Christian C.

doi:10.1002/0471142301.ns0715s30

Cited by 5 publications

(2 citation statements)

References 83 publications

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“…One conundrum that we have faced in identifying G protein-coupled pathways is that the [ 35 S]GTPɣS-binding assay provides a net sum of all G proteins activated and is influenced by the rates at which the GDP is able to dissociate from the various Gα proteins. Identification of the relative activation of coupled Gα proteins can be determined via [ 35 S]GTPɣS binding using an antibody-targeted GTPɣS scintillation proximity assay (SPA) (Delapp et al, 1999; Kahl & Felder, 2005), which quantitates [ 35 S]GTPɣS binding to each G protein individually. This procedure can mitigate concerns related to activation or inhibition of multiple G proteins and reduces the variability in sensitivity by different G proteins (Milligan, 2003; Strange, 2010).…”

Section: Introductionmentioning

confidence: 99%

Mouse Neuroblastoma CB1 Cannabinoid Receptor-Stimulated [35S]GTPɣS Binding: Total and Antibody-Targeted Gα Protein-Specific Scintillation Proximity Assays

Eldeeb

Leone-Kabler

Howlett

2017

Methods in Enzymology

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G protein-coupled receptors (GPCRs) are important regulators of cellular signaling functions and therefore are a major target for drug discovery. The CB cannabinoid receptor is among the most highly expressed GPCRs in neurons, where it regulates many differentiated neuronal functions. One model system for studying the biochemistry of neuronal responses is the use of neuroblastoma cells originating from the C1300 tumor in the A/J mouse, including cloned cell lines NS20, N2A, N18TG2, N4TG1, and N1E-115, and various immortalized hybrids of neurons with N18TG2 cells. GPCR signal transduction is mediated through interaction with multiple types and subtypes of G proteins that transduce the receptor stimulus to effectors. The [S]GTPɣS assay provides a valuable pharmacological method to evaluate efficacy and potency in the first step in GPCR signaling. Here, we present detailed protocols for the [S]GTPɣS-binding assay to measure the total G protein binding and the antibody-targeted scintillation proximity assay to measure specific Gα proteins in neuroblastoma cell membrane preparations. This chapter presents step-by-step methods from cell culture, membrane preparation, assay procedures, and data analysis.

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Section: Introductionmentioning

confidence: 99%

Mouse Neuroblastoma CB1 Cannabinoid Receptor-Stimulated [35S]GTPɣS Binding: Total and Antibody-Targeted Gα Protein-Specific Scintillation Proximity Assays

Eldeeb

Leone-Kabler

Howlett

2017

Methods in Enzymology

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“…Although the research work with a use of this technique has provided us with invaluable results regarding physiological, pharmacological, and pathological functionality of G protein-mediated signal transduction stimulated by many metabotropic receptors, it is usually applicable preferentially to G i/o proteins, through which the inhibitory receptors are negatively coupled with adenylyl cyclase, especially in native brain membranes. More recently, the antibody-capture [ 35 S]GTPcS scintillation proximity assay (SPA), in which immuno-capture of Ga subunits following [ 35 S]GTPcS binding is combined with the SPA technology (Kahl and Felder 2005), has been newly developed to investigate the specific interactions between several G protein-coupled receptors (GPCRs) and Ga subunits, even in native brain membranes (DeLapp 2004). Using the membranes prepared from rodent brain regions, functional activation of Ga subunits have been reported for Ga q/11 , Ga i(1-3) , and Ga o coupled with muscarinic acetylcholine receptors (mAChRs) in rat striatum (DeLapp et al 1999), Ga q/11 coupled with M 1 mAChR in mouse hippocampus and cortex (Porter et al 2002), Ga o and Ga i3 in rat hippocampus and Ga i3 in rat anterior raphe coupled with 5-HT 1A receptors (Mannoury la Cour et al 2006), Ga s/olf and Ga q coupled with dopamine D 1 receptors in rat striatum and cortex (Mannoury la Cour et al 2007), Ga o coupled with 5-HT 1A receptors in rat hippocampus (Martel et al 2007), and Ga o and Ga i1/3 coupled with GABA B receptors in rat cortex, hippocampus, and cerebellum (Mannoury la Cour et al 2008).…”

Section: Introductionmentioning

confidence: 99%

Muscarinic acetylcholine receptor-mediated activation of Gq in rat brain membranes determined by guanosine-5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding using an anti-G protein scintillation proximity assay

Odagaki

Toyoshima

2011

J Neural Transm

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In the present study, we performed antibody-capture guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) scintillation proximity assay (SPA), in which immuno-capture of Gα subunits following [(35)S]GTPγS binding was combined with SPA technology, in rat brain membranes. Preliminary experiments using a series of agonists and commercially available anti-Gα antibodies indicated the increase in specific [(35)S]GTPγS binding to Gα(q) determined with the anti-Gα antibody sc-393 and evoked by carbamylcholine chloride (CCh) was pharmacologically relevant. The experimental conditions were optimized as for the concentrations of GDP, MgCl(2), and NaCl, the dilution of the anti-Gα(q) antibody, and membrane protein contents incubated. Under the optimized conditions, CCh-stimulated specific [(35)S]GTPγS binding to Gα(q) in a concentration-dependent and saturable manner with an EC(50) of around 10 μM in all of the membranes prepared from rat hippocampus, cerebral cortex, and striatum. The maximum responses were varied according to the brain regions, with the rank order in magnitude of hippocampus > cerebral cortex > striatum. The addition of MT-7, a snake toxin with high selectivity for M(1) over the other muscarinic acetylcholine receptors (mAChRs) (M(2)-M(5)), almost completely extinguished CCh-stimulated [(35)S]GTPγS binding to Gα(q), even at a concentration as low as 1 nM. These results indicate that the functional coupling between M(1) mAChR and Gα(q) can be investigated in rat native brain membranes by means of antibody-capture SPA/[(35)S]GTPγS binding assay. The assay developed in the present study would provide a useful strategy for investigation of possible pathophysiological alterations in neuropsychiatric disorders such as Alzheimer's disease and schizophrenia as well as for drug discovery.

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Deuterium‐ und tritiummarkierte Verbindungen: Anwendungen in den modernen Biowissenschaften

Atzrodt¹,

Derdau²,

Kerr

et al. 2018

Angewandte Chemie

108

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Wasserstoffisotope sind einzigartige Werkzeuge zur Identifizierung und Erforschung biologischer oder chemischer Vorgänge. Wasserstoffisotopenmarkierungen erlauben den spurlosen und direkten Einbau einer zusätzlichen Masseneinheit oder eines radioaktiven Markers in ein organisches Molekül, und dies praktisch ohne Änderungen der chemischen Struktur, der physikalischen Eigenschaften oder der biologischen Aktivität. Die Verwendung von deuteriummarkierten Isotopologen zur Untersuchung der spezifischen massenspektrometrischen (MS) Muster, die von Gemischen biologisch relevanter Moleküle hervorgerufen werden, ermöglicht eine drastische Vereinfachung der Analyse. Solche Methoden ermöglichen nun tiefe Einblicke in einen großen, kontinuierlich anwachsenden Bereich von Anwendungen in den Biowissenschaften und darüber hinaus. Speziell Tritium (3H) wird zunehmend eingesetzt, insbesondere in der pharmazeutischen Wirkstoffsuche. Aufwand und Kosten der Synthese markierter Verbindungen werden durch die hohe Empfindlichkeit der Analyse und hohe Zuverlässigkeit der erhaltenen Daten mehr als ausgeglichen. In diesem Aufsatz werden Fortschritte bei der Verwendung von Wasserstoffisotopen in den Biowissenschaften beschrieben.

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Scintillation Proximity Assay

Cited by 5 publications

References 83 publications

Mouse Neuroblastoma CB1 Cannabinoid Receptor-Stimulated [35S]GTPɣS Binding: Total and Antibody-Targeted Gα Protein-Specific Scintillation Proximity Assays

Mouse Neuroblastoma CB1 Cannabinoid Receptor-Stimulated [35S]GTPɣS Binding: Total and Antibody-Targeted Gα Protein-Specific Scintillation Proximity Assays

Muscarinic acetylcholine receptor-mediated activation of Gq in rat brain membranes determined by guanosine-5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding using an anti-G protein scintillation proximity assay

Deuterium‐ und tritiummarkierte Verbindungen: Anwendungen in den modernen Biowissenschaften

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