3h muscimol

Manufactured by PerkinElmer

Sourced in United States

[3H]Muscimol is a radioactive chemical compound used in research applications. It serves as a ligand for the GABA-A receptor, a type of receptor for the neurotransmitter gamma-aminobutyric acid (GABA). This product can be utilized in various experimental settings to study GABA-mediated signaling and related biological processes.

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11 protocols using 3h muscimol

GABA Receptor Binding Assay with [3H]Muscimol

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In brief, binding of [ 3 H]muscimol (specific activity: 22.46 Ci/mmol, PerkinElmer, USA) to GABA A receptors using [ 3 H]muscimol was performed based on procedures previously described (Deng and Huang, 2006; Ling and Caspary, 2013) . In brief, sections were preincubated in 50 mM Tris-Citrate buffer (pH=7.0), three times each for 5 min at 4 ºC. Sections were then incubated for 45 min at 4 ºC in the same buffer containing 3 nM [ 3 H]muscimol (specific activity: 22.46 Ci/mmol; Perkin-Elmer, USA) for the total binding. Non-specific binding was determined with the addition of 100 mΜ GABA. After incubation, the sections were washed in ice-cold buffer (4 × 2 s), dipped in distilled water and air dried (Deng and Huang, 2006; Ling and Caspary, 2013) .

Lian J, & Deng C. (2019). Early antipsychotic exposure affects NMDA and GABAA receptor binding in the brains of juvenile rats. Psychiatry research, 273.

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Characterization of Cedarwood Oil Using GC-MS

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CO was purchased from Thai-China Flavors and Fragrances Industry (Nonthaburi, Thailand). The obtained CO was analyzed using gas chromatography–mass spectrometry (GC-MS) and the method previously described [23 (link)]. Nile Red, polysorbate 20, Kolliphor EL, methanol, acetonitrile, GABA, diazepam, eugenol, and dimethyl sulfoxide (DMSO) were from Sigma-Aldrich (St. Louis, MO, USA). Captex 300 and Capmul MCM EP were kindly gifted from Abitec (Columbus, OH, USA). Ethanol and isopropanol were from VWR International (Radnor, PA, USA). Purified water was obtained from a Millipore Milli-Q Ultrapure Water purification system (Billeria, MA, USA). [³H]Muscimol (28.5 Ci/mmol) was from PerkinElmer (Waltham, MA, USA).

Kheawfu K., Pikulkaew S., Wellendorph P., Jørgensen L.V., Rades T., Müllertz A, & Okonogi S. (2022). Elucidating Pathway and Anesthetic Mechanism of Action of Clove Oil Nanoformulations in Fish. Pharmaceutics, 14(5), 919.

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Radioligand Binding Assay for GABA Receptors

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[³H]muscimol binding assays were performed using a previously described method with minor modification [59 (link)]. Briefly, HEK cell membranes proteins (50 μg/ml final concentration) were incubated with 1–2 nM [³H]muscimol (30 Ci/mmol; PerkinElmer Life Sciences), neurosteroid in different concentrations (1 nM-10 μM), binding buffer (10 mM potassium phosphate, 100 mM KCl [pH 7.5]), in a total volume of 1 ml. Assay tubes were incubated for 1 hour at 4°C in the dark. Nonspecific binding was determined by binding in the presence of 1 mM GABA. Membranes were collected on Whatman/GF-C glass filter paper using a Brandel cell harvester (Gaithersburg, MD). To determine the Bmax of [³H]muscimol binding, 100 μg/ml of proteins were incubated with 250 nM [³H]muscimol, with specific activity reduced to 2 Ci/mmol, for 1 hour at 4°C in the dark. The membranes were collected on Whatman/GF-B glass filter papers using manifold. Radioactivity bound to the filters was measured by liquid scintillation spectrometry using Bio-Safe II (Research Products International Corporation). Each data point was determined in triplicate.

Chen Z.W., Bracamontes J.R., Budelier M.M., Germann A.L., Shin D.J., Kathiresan K., Qian M.X., Manion B., Cheng W.W., Reichert D.E., Akk G., Covey D.F, & Evers A.S. (2019). Multiple functional neurosteroid binding sites on GABAA receptors. PLoS Biology, 17(3), e3000157.

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High-throughput screening of GABA receptor modulators

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Dulbecco’s modified Eagle medium (DMEM) containing GlutaMAX-I, foetal bovine serum (FBS), Dulbecco’s phosphate-buffered saline (DPBS), penicillin-streptomycin, hygromycin B, trypsin-EDTA, and Hank’s balanced salt solution (HBSS) were all purchased from Life Technologies (Paisley, UK). Polyfect transfection reagent was purchased from Qiagen (West Sussex, UK). FLIPR membrane potential Blue dye was purchased from Molecular Devices (Crawley, UK). Buffer components, poly-D-lysine (PDL) and 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES) were purchased from Sigma-Aldrich (St. Louis, MO, USA). GABA and gabazine were from Tocris Bioscience (Bristol, UK). Compounds in the screening library and analogues were purchased from either Enamine, Vita-M or ChemBridge as stated in the supplementary information. [³H]muscimol (36.9 Ci/mmol) was purchased from PerkinElmer (Waltham, MA, USA). Compounds 2027 and 018 can be purchased from Enamine and have ID numbers Z1839922409 and Z1839935473, respectively.

Falk-Petersen C.B., Tsonkov T.M., Nielsen M.S., Harpsøe K., Bundgaard C., Frølund B., Kristiansen U., Gloriam D.E, & Wellendorph P. (2020). Discovery of a new class of orthosteric antagonists with nanomolar potency at extrasynaptic GABAA receptors. Scientific Reports, 10, 10078.

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Receptor Binding Density Assay Protocol

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The receptor binding density for AMPA, NMDA, and GABA_A receptors was adapted from the protocols described in (24 (link)). The tritiated ligands [³H]AMPA, [³H]MK-801, and [³H]Muscimol were purchased from PerkinElmer (Germany). AMPA is an agonist of the homonymous receptor, MK-801, also known as dizocilpine, is an uncompetitive antagonist of the NMDA receptor and Muscimol is an agonist of the GABA_A receptor. In the first step, the pre-incubation, endogenous ligands were washed off. In the following main incubation, the tritiated ligands were incubated both in the presence of a competitor, in order to determine the unspecific binding, and without it, in order to assess the total binding. Finally, the slices were rinsed. The slices incubated with [³H]AMPA were additionally dried with a warm air stream for 2 s and afterwards with a cold air stream. Slices incubated with [³H]MK-801 and [³H]Muscimol were dried with a cold air stream. A detailed description of the protocols used is reported in Table 1.

Nardi L., Chhabra S., Leukel P., Krueger-Burg D., Sommer C.J, & Schmeisser M.J. (2023). Neuroanatomical changes of ionotropic glutamatergic and GABAergic receptor densities in male mice modeling idiopathic and syndromic autism spectrum disorder. Frontiers in Psychiatry, 14, 1199097.

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Radioactive Ligand Binding Assay

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Radioactive [³H]-flunitrazepam (Methyl-[³H], 81.4 Ci/mmol), [³H]-muscimol (Methylene-[³H], 25.5 Ci/mmol), and [³H]-spiperone (Benzene ring-[³H], 15.7 Ci/mmol) were obtained from Perkin-Elmer (Life Sciences, Inc., Boston, MA). Dilutions were made using 50 mM Tris-Cl, pH 7.4, and they were stored at 4°C. Diazepam (DZ) and GABA were obtained from Sigma (St. Louis, MO, USA). Diazepam was dissolved in 0.9% NaCl in the presence of 1% DMSO for animal tests. These drugs were administered to mice in a delivery volume of 10 ml/kg through the oral route. d-galactose, malondialdehyde (MDA), and thiobarbituric acid (TBA) were obtained from Sigma (St. Louis, MO, USA), and d-galactose was dissolved in 0.9% NaCl for animal treatment.

Li R., Chan W., Mat W., Ho Y., Yeung R.K., Tsang S, & Xue H. (2017). Antiaging and Anxiolytic Effects of Combinatory Formulas Based on Four Medicinal Herbs. Evidence-based Complementary and Alternative Medicine : eCAM, 2017, 4624069.

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Radioligand Binding Assay Protocols

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[³H] muscimol (36.9-38.3 Ci/mmol) was purchased from Perkin Elmer, Inc. (Waltham, MA, United States), while [³H] CGP54626 (30 Ci/mmol) was purchased from American Radiolabeled Chemicals, Inc. (St. Lousi, MO, United States).

Pehrson A.L., Pedersen C.S., Tølbøl K.S, & Sanchez C. (2018). Vortioxetine Treatment Reverses Subchronic PCP Treatment-Induced Cognitive Impairments: A Potential Role for Serotonin Receptor-Mediated Regulation of GABA Neurotransmission. Frontiers in Pharmacology, 9, 162.

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Purification and Characterization of Human GABAARs

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4-Bromo-2,6-diisopropylaniline and anhydrous grade solvents used in synthesis were from Aldrich, and were not further dried or purified. Common chemicals, etomidate, asolectin, FLAG peptide and polyethyleneimine were from Sigma. Buffer chemicals, CHAPS and DDM were from Fisher–Anatrace. pTFD-BnOH was obtained from TCI America. R–mTFD-MPAB, [³H]R–mTFD-MPAB (38 Ci/mmol, 26 μM in ethanol, and [³H]azietomidate (19 Ci/mmol, 53 μM in ethanol) were synthesized and tritiated previously [22 (link), 32 (link)]. [³H]Muscimol and [³H]flunitrazepam were from Perkin Elmer (Cat. # NET 574 250UC and NET 567250UC respectively).
The human GABA_ARs used for the biochemical and photolabeling studies described herein and designated as α1β3γ2L or α1β3 had the composition N-FLAG–α1β3γ2L–C–(GGS)3GK–1D4 or N-FLAG–α1β3 respectively and were expressed in tetracycline-inducible HEK293 cells as previously described [33 (link), 34 (link)]. They were used as native membranes or after solubilization and purification on a FLAG antibody column, from which they were eluted in micelles of 200 μM asolectin and 5 mM CHAPS with 100 μg/mL (~100 μM) FLAG peptide. Membranes and reconstituted receptors were stored at (–80°) until needed. In electrophysiological studies, the human subunits lacked the purification tags (see below).

Shalabi A.R., Yu Z., Zhou X., Jounaidi Y., Chen H., Dai J., Kent D.E., Feng H.J., Forman S.A., Cohen J.B., Bruzik K.S, & Miller K.W. (2020). A Potent Photoreactive General Anesthetic with Novel Binding Site Selectivity for GABAA Receptors.. European journal of medicinal chemistry, 194, 112261.

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Radiolabeled Ligand Binding Assay

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[³H]Muscimol (22 Ci/mmol) and [³H]EBOB (48 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA, USA). [³H]GABA (30 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA, USA). Unlabeled GABA and picrotoxin were from Sigma Chemicals Co. (St. Louis, MO, USA). AA29504 and THIP were from Tocris Biosciences (Bristol, UK).

Sikstus S., Benkherouf A.Y., Soini S.L, & Uusi-Oukari M. (2021). The Influence of AA29504 on GABAA Receptor Ligand Binding Properties and Its Implications on Subtype Selectivity. Neurochemical Research, 47(3), 667-678.

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GABAA Receptor Binding Density Assay

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GABAA receptor binding density was examined based on methods previously described by our laboratory and others (du Bois et al., 2009; (link)Xia and Haddad, 1992; Yu et al., 2013) (link).
Briefly, sections were pre-incubated three times in 50 mM Tris-citrate (pH 7.0) buffer at 4ºC for 5 mins. Sections were incubated in 50 mM Tris-citrate buffer containing 50 nM [ 3 H] muscimol (specific activity 29.5 Ci/mmol, PerkinElmer, Boston, USA), a selective agonist for the GABAAR, for 45 min at 4ºC. Non-specific binding was determined by incubating sections in 50 mM Tris-citrate (pH 7.0) buffer containing [ 3 H] muscimol and 100 µM GABA (Sigma-Aldrich, Castle Hill, NSW, Australia). After incubation, sections were rinsed four times for 2 sec each in 50 mM Tris-citrate buffer (4°C), rinsed in cold milliQ water and airdried overnight.

Osborne A.L., Solowij N., Babic I., Lum J.S., Newell K.A., Huang X.F, & Weston-Green K. (2019). Effect of cannabidiol on endocannabinoid, glutamatergic and GABAergic signalling markers in male offspring of a maternal immune activation (poly I:C) model relevant to schizophrenia. Progress in neuro-psychopharmacology & biological psychiatry, 95.

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