3h pentazocine

Manufactured by PerkinElmer

Sourced in Belgium, United Kingdom

[3H](+)-pentazocine is a radioactive compound used as a research tool for studying the sigma-1 receptor. It has a high affinity and selectivity for the sigma-1 receptor, making it a valuable tool for researchers investigating the role of this receptor in various biological processes.

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Lab products found in correlation

Haloperidol, by Merck Group (3 mentions) 3h dtg, by PerkinElmer (3 mentions) Trypsin, by Thermo Fisher Scientific (2 mentions) Phenytoin, by Merck Group (2 mentions) Pentazocine, by PerkinElmer (2 mentions) Ultima gold mv scintillation cocktail, by PerkinElmer (2 mentions) 3h 1 3 di o tolylguanidine 3h dtg, by PerkinElmer (2 mentions) Pentazocine, by Merck Group (2 mentions) Haloperidol, by Bio-Techne (2 mentions) Dextromethorphan, by Merck Group (1 mentions) Prostaglandin e2 pge2, by Cayman Chemical (1 mentions) Probdnf, by Alomone (1 mentions) 4 ppbp, by Bio-Techne (1 mentions) Pf 04418948, by Bio-Techne (1 mentions) Net1056, by PerkinElmer (1 mentions) Net986, by PerkinElmer (1 mentions) Bd1063, by Bio-Techne (1 mentions) C57bl 6j male mice, by Charles River Laboratories (1 mentions) Hek293 cell, by American Type Culture Collection (1 mentions) Microbeta microplate counter, by PerkinElmer (1 mentions)

Spelling variants of this product

((+)-Pentazocine (2 citations)

21 protocols using 3h pentazocine

Radio-Labeled Ligand Preparation

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Tissue culture media, antibiotics, trypsin, and serum were purchased from Invitrogen (Paisley, UK) or Sigma-Aldrich (Ireland). [³H] (+) pentazocine ((1S,9S,13S)-1,13-dimethyl-10-(3-methylbut-2-en-1-yl)-10-azatricyclotrideca-2,4,6-trien-4-ol) and [5-³H(N)]- 1,3-di-O-tolylguanidine (DTG) were purchased from PerkinElmer (Beaconsfield, UK). Other reagents were purchased from Sigma-Aldrich (Poole, UK). Before use, drugs were dissolved in an appropriate vehicle and diluted in assay buffer. The pH of each solution was adjusted to 7.4.

Abbas H., Borde P., Willars G.B., Ferry D.R, & Safrany S.T. (2020). Hazards of Using Masking Protocols When Performing Ligand Binding Assays: Lessons From the Sigma-1 and Sigma-2 Receptors. Frontiers in Pharmacology, 11, 309.

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Sigma-1 Receptor Ligand Binding Protocol

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Functional profile of compound 1 was obtained by using the phenytoin method which uses the same protocol as S1R radioligand binding assay with some differences. The experiments were performed one time in the presence of phenytoin and one time without phenytoin. In both cases the final volume was 0.5 mL and it was composed as follows: 200 μL of membrane preparation, 50 μL of 20 nM [³H](+)-pentazocine (28.4 Ci mmol⁻¹, PerkinElmer), 50 μL of cold ligand or its solvent, 180 μL of Tris buffer (50 mM, pH 8) and 20 μL of 25 mM phenytoin (Merck Life Science S.r.l.) or its solvent (0.3 M NaOH). The incubation of samples lasts 120 min at 37 °C. Unlabeled (+)-pentazocine (10 μM) was used to measure non-specific binding. The molecules are defined S1R agonist if the K_i ratio without/with phenytoin is >1 and antagonist if the K_i ratio without/with phenytoin is ≤1.^{24 (link)}

De Luca L., Lombardo L., Mirabile S., Marrazzo A., Dichiara M., Cosentino G., Amata E, & Gitto R. (2023). Discovery and computational studies of piperidine/piperazine-based compounds endowed with sigma receptor affinity. RSC Medicinal Chemistry, 14(9), 1734-1742.

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S1R Receptor Binding Assay with WLB-73502

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Guinea pig brain membrane binding assays for the S1R (σ₁R) using [³H](+)-pentazocine (PerkinElmer) as radioligand were conducted either in the absence or presence of 1 mmol/L phenytoin (DPH) (Sigma–Aldrich), as previously described²⁹ (link), to identify the functional (agonistic or antagonistic) nature of WLB-73502. Dextromethorphan (Sigma–Aldrich) and haloperidol (Sigma–Aldrich) were used as control, prototypical S1R agonist and antagonist, respectively.

Vidal-Torres A., Fernández-Pastor B., García M., Ayet E., Cabot A., Burgueño J., Monroy X., Aubel B., Codony X., Romero L., Pascual R., Serafini M.T., Encina G., Almansa C., Zamanillo D., Merlos M, & Vela J.M. (2022). Bispecific sigma-1 receptor antagonism and mu-opioid receptor partial agonism: WLB-73502, an analgesic with improved efficacy and safety profile compared to strong opioids. Acta Pharmaceutica Sinica. B, 13(1), 82-99.

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Compound Sourcing and Preparation for Pharmacological Studies

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Compounds were purchased from the following sources: BD1063, 4-PPBP, and PF-04418948 from Tocris Biosciences (Minneapolis, MN); Haloperidol from Santa Cruz Biotechnologies, Inc. (Dallas, TX); Haloperidol metabolites I and II (4-(4-Chlorophenyl)-4-hydroxypiperidine and (±)-4-(4-Chlorophenyl)-α-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol, respectively) from Sigma-Aldrich (St. Louis, MO); Prostaglandin E₂ (PGE₂) from Cayman Chemical (Ann Arbor, MI). Radioligands were purchased from PerkinElmer: ([³H]-(+)-pentazocine ((+)-Pentazocine, [RING-1,3-³H], 33.9 Ci/mmol, NET1056), [³H]DTG (1,3-Di-o-tolylguanidine, [p-RING-³H]-, 50 Ci/mmol, NET986) (Saint Louis, MO). With the exception of PGE₂, which was dissolved in ethanol:water (1:1 v/v), stock concentrations of all compounds were prepared in DMSO at concentrations ranging from 10–100 mM. Stocks were then diluted 1:1000 (v/v) in the final assay solution. ProBDNF was from Alomone Labs (Cat. No. B-257, Jerusalem, Israel) and the source of the mature BDNF was from the BDNF standard provided in the Emax ImmunoAssay kit (Cat. No. G7611, Promega, Madison, WI).

Dalwadi D.A., Kim S, & Schetz J.A. (2017). Activation of the Sigma-1 receptor by haloperidol metabolites facilitates brain-derived neurotrophic factor secretion from human astroglia. Neurochemistry international, 105, 21-31.

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Male Mice Receptor Binding Assay

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CD-1 and C57BL/6J male mice at 6–8 weeks of age were obtained from Charles River Laboratories and Jackson Laboratory, respectively. [³H] (+)-Pentazocine was purchased from PerkinElmer (Boston, MA). HEK293 cells were obtained from ATCC (Manassas, VA). All oligonucleotides were synthesized and purified by Sigma-Aldrich (St. Louis, MO). All other materials were obtained from the sources listed.

Pan L., Pasternak D.A., Xu J., Xu M., Lu Z., Pasternak G.W, & Pan Y.X. (2017). Isolation and characterization of alternatively spliced variants of the mouse sigma1 receptor gene, Sigmar1. PLoS ONE, 12(3), e0174694.

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Membrane Fraction Binding Assay for σ1 Receptor

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Membrane fraction of HEK293T Δσ1R cells was prepared as previously described (Yano et al., 2018 (link)). The radioligand incubation was carried out in 96-well plates containing 60 μL fresh Earle’s Balanced Salts Solution (EBSS) binding buffer (8.7 g/l Earle’s Balanced Salts without phenol red (United States Biological) and 2.2 g/L sodium bicarbonate, pH to 7.4), 20 μL of (+)-pentazocine (varying concentrations), 100 μL membranes (25 μg/well total protein), and 20 μL of radioligand diluted in binding buffer (1 nM [³H]-(+)-pentazocine (American Radiolabeled Chemicals), final concentration) for each well. We used 1 mM (+)-pentazocine as a non-specific control. Concentrations for non-radioactive (+)-pentazocine in homologous competition assay were: 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1 nM, and 0.1 nM in EBSS with 10% DMSO. Total binding was determined with 1% DMSO vehicle (final concentration). All compound dilutions were tested in triplicate, and samples were incubated for 120 min at room temperature. The reactions were terminated by filtration through PerkinElmer Uni Filter-96 GF/B, presoaked in 0.05% PEI for 120 min, and the 96-well filter plates were counted in PerkinElmer MicroBeta Microplate Counter as described (Yano et al., 2018 (link)) with counter efficiency at 31% for [³H]-(+)-pentazocine. K_d and B_max values were determined from at least three independent experiments.

Yano H., Liu L., Naing S, & Shi L. (2019). The Effects of Terminal Tagging on Homomeric Interactions of the Sigma 1 Receptor. Frontiers in Neuroscience, 13, 1356.

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Receptor Binding Assays for S1R and S2R

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Brain and liver homogenates for S1R and S2R receptor binding assays were prepared from male Dunkin-Hartley guinea pigs and Sprague Dawley rats, respectively (ENVIGO RMS S.R.L., Udine, Italy; Italian Minister of Health, authorization for animal experimentation—Project acronym 335/1984F.N.JLT). [³H](+)-pentazocine (26.9 Ci/mmol) and [³H]1,3-di-o-tolylguanidine ([³H]DTG, 35.5 Ci/mmol) were purchased from PerkinElmer (Zaventem, Belgium). Ultima Gold MV Scintillation cocktail was from PerkinElmer (Milan, Italy). All the other materials were obtained from Merck Life Science S.r.l. (Milan, Italy). UV absorbance was measured using a microplate spectrophotometer reader (Synergy HT, Biotec). The bound radioactivity has been determined using a Beckman LS 6500 liquid scintillation counter (Beckman Coulter, Brea, CA, USA).

Zampieri D., Calabretti A., Romano M., Fortuna S., Collina S., Amata E., Dichiara M., Marrazzo A, & Mamolo M.G. (2023). Cytotoxicity Profiles and Neuroprotective Properties of the Novel Ifenprodil Analogues as Sigma Ligands. Molecules, 28(8), 3431.

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Radioligand Binding Assay for σ1 Receptor

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Radioligand binding experiments were performed similarly to established procedures^{23 (link)}. In brief, Sf9 membranes expressing σ₁ receptor were prepared by dounce homogenization followed by centrifugation. Resuspended membranes were aliquoted and flash frozen prior to use. For each binding experiment, membranes were incubated with ³H (+)-pentazocine (Perkin Elmer) at the indicated concentration or at a fixed concentration of 10 nM for competition binding assays. To approximate physiological conditions, incubation was carried out at 37 °C for 2 hours in 150 mM sodium chloride and 20 mM HEPES pH 7.5. Filter pads were incubated with 0.3% polyethyleneimine for 20 minutes, then samples were loaded onto the filter and washed using a Brandel harvester. Radioactivity was quantified by liquid scintillation counting. Non-specific binding was quantified by replicate reactions in the presence of 2 μM haloperidol. All measurements were performed in triplicate and repeated in two independent experiments. Experiments in Tris pH 7.5 showed similar results to those conducted in HEPES. Data analysis was performed in GraphPad Prism, with K_i values calculated by Cheng-Prusoff correction using the experimentally measured probe K_d.

Schmidt H.R., Zheng S., Gurpinar E., Koehl A., Manglik A, & Kruse A.C. (2016). Crystal structure of the human σ1 receptor. Nature, 532(7600), 527-530.

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Radioligand Binding Assay Protocols

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(−)-Cocaine hydrochloride, haloperidol, (+)-pentazocine, 1,3-di(2-tolyl)guanidine (DTG), dextromethorphan hydrobromide, (+)- and (−)-N-allylnormetazocine (NANM, SKF10047), ifenprodil (+)-tartrate, BD1063 (1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine) dihydrochloride, and GBR12909 dihydrochloride were obtained from Sigma-Aldrich, Inc. (St. Louis, MO) or Tocris Bioscience (Minneapolis, MN). [³H]DTG (48 – 53 Ci/mmol) and [³H](+)-pentazocine (35 – 37 Ci/mmol) were obtained from PerkinElmer, Inc. (Waltham, MA). E-N-1-(3′-iodoallyl)-N′-4-(3″,4″-dimethoxyphenethyl)-piperazine (E-IA-DM-PE-PIPZE) and [¹²⁵I]E-IA-DM-PE-PIPZE (ca. 2000 Ci/mmol) were prepared as previously described (Lever et al., 2012 (link)). [¹²⁵I]RTI-121 (3β-(4-iodophenyl)tropan-2β-carboxylic acid isopropyl ester) was prepared (ca. 2000 Ci/mmol) as previously described (Lever et al., 1996 (link)). Sterile bacteriostatic saline (0.9% NaCl, 0.9% benzyl alcohol; w/v) was used in formulations for animal studies. Other chemicals and solvents were the best available commercial grade, and were used as received.

LEVER J.R., FERGASON-CANTRELL E.A., WATKINSON L.D., CARMACK T.L., LORD S.A., XU R., MILLER D.K, & LEVER S.Z. (2015). Cocaine Occupancy of Sigma1 Receptors and Dopamine Transporters in Mice. Synapse (New York, N.Y.), 70(3), 98-111.

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Pentazocine Binding Kinetics Assay

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Membrane samples prepared as described above were thawed, syringe homogenized, and diluted in 50 mM Tris pH 8.0 to a final concentration of 0.05 mg/mL in a 96-well plate with a final volume of 100 μL per well. For association experiments, samples were incubated with 1 nM, 10 nM, or 100 nM [³H](+)-pentazocine for 5 min to 6 h, with all points performed in triplicate. For dissociation experiments, samples were first incubated with 10 nM [³H](+)-pentazocine (Perkin Elmer) for 90 minutes at 37 °C to reach equilibrium. After equilibration, 1 μL 500 μM haloperidol (Tocris Biosciences) was added to a set of wells in triplicate for a particular time point. This was repeated for a total of eight time points over the course of twenty-four hours. Upon completion of the time course in either the association or dissociation experiment, the reaction was terminated by massive dilution in ice-cold water and filtration over a glass microfiber filter using a Brandel harvester. Filters were soaked in 0.3% PEI for at least 30 minutes prior to use. Radioactivity was quantified by liquid scintillation counting. Data analysis was performed using GraphPad Prism.

Schmidt H.R., Betz R.M., Dror R.O, & Kruse A.C. (2018). Structural basis for σ1 receptor ligand recognition. Nature structural & molecular biology, 25(10), 981-987.

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