TAKEM4 buffer: 50 mM Tris–HCl, pH 7.5, 70 mM NH4Cl, 30 mM KCl, 1 mM EDTA and 4 mM MgCl2. [C5–3H]-UTP used in radioactive in vitro transcriptions was purchased from Moravek. Q5 DNA polymerase was purchased from New England Biolabs (NEB). Ecolite™ scintillation cocktail was purchased from MP Biomedicals. All other enzymes and chemicals were obtained from Fisher Scientific.
Ecolite scintillation cocktail
Manufactured by MP Biomedicals
Sourced in United States
Ecolite Scintillation cocktail is a liquid scintillation counting solution designed for the detection and measurement of radioactivity. It is a clear, homogeneous solution composed of organic solvents, surfactants, and fluorescent compounds. The core function of Ecolite Scintillation cocktail is to convert the energy of radioactive emissions into light, which can then be detected and quantified by a liquid scintillation counter.
Automatically generated - may contain errors
Lab products found in correlation
Ls6000 scintillation counter, by Beckman Coulter (6 mentions)
Formic acid, by Thermo Fisher Scientific (3 mentions)
96 well low binding plates, by Greiner (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Q5 dna polymerase, by New England Biolabs (1 mentions)
3h leucine, by MP Biomedicals (1 mentions)
Tri carb 2100tr liquid scintillation analyzer, by Hewlett-Packard (1 mentions)
3h thymidine, by MP Biomedicals (1 mentions)
Tris base, by Bio-Rad (1 mentions)
Dextran 70, by Merck Group (1 mentions)
Triton x 100, by Bio-Rad (1 mentions)
Mn chloride tetrahydrate mncl2 4h2o, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (1 mentions)
Radioactive 14c sucrose, by Moravek Biochemicals (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Homovanillic acid, by Merck Group (1 mentions)
Taxifolin, by Merck Group (1 mentions)
Isovanillin, by Merck Group (1 mentions)
17 protocols using ecolite scintillation cocktail
1
In Vitro Transcription Protocol
2
Radioligand Binding Assay for Ryanodine Receptors
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[3H]-ryanodine binding assays were performed as outlined previously (Fruen et al., 2000 (link)). Briefly, in 96-well plates, HSR vesicles (1 mg/mL) and cardiac CSR vesicles (3mg/mL) were incubated with 0.02% DMSO or compound (concentration indicated) for 3hr at 37°C in a solution containing 150mM KCl, 0.1μM calmodulin, 5 mM GSH, 2mM dithiothreitol, 1 μg/mL Aprotinin and Leupeptin, 1 mM EGTA, 238μM or 1.62mM CaCl2 (as determined by MaxChelator to yield 100 nM or 30 μM of free Ca2+, respectively), 0.1 mg/mL BSA, [3H]-ryanodine (7.5 and 10nM for cardiac and skeletal SR, respectively) and 20mM K-PIPES (pH 7.0). Non-specific and maximal [3H]-ryanodine binding to SR were separately assessed by addition of 40μM non-radioactively labeled ryanodine or 5mM Adenylyl-imidodiphosphate, respectively. Such control samples were each distributed over 4 wells/plate. After incubation, unbound [3H]-ryanodine was removed by filtration through grade GF/B Glass Microfiber filters (Brandel Inc., Gaithersburg, MD) using a 96-sample Brandel Harvester. In 4mL of Ecolite Scintillation cocktail (MP biomedicals, Solon, OH, USA), Radioactivity on the filter was counted using a Beckman LS6000 scintillation counter (Fullerton, CA). All experiments were run with 3 different batches of SR. Statistical analysis was performed using a two-way, unpaired Student's T-test.
3
Ryanodine Receptor Binding Assay
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In 96-well plates, skeletal SR membranes (HSR, 1 mg/ml) and cardiac SR membranes (CSR, 3 mg/ml) were preincubated with 1% DMSO or hit compound for 30 min at 22 °C in a solution containing 150 mM KCl, 5 mM GSH, 1 μg/ml Aprotinin/Leupeptin, 1 mM EGTA, and 65 μM or 1.02 mM CaCl2 (as determined by MaxChelator to yield 30 nM or 30 μM of free Ca2+, respectively), 0.1 mg/ml of BSA, and 20 mM K-PIPES (pH 7.0). Nonspecific [3H]ryanodine binding to SR was assessed by addition of 40 μM nonradioactively labeled ryanodine. Maximal [3H]ryanodine binding was assessed by addition of 5 mM adenylyl-imidodiphosphate (AMP-PNP), and in the case of CSR, 20 mM caffeine was added. These control samples were each loaded over four wells per plate. Binding of [3H]ryanodine (7.5 and 10 nM for CSR and HSR, respectively) was determined following a 3 h incubation at 37 °C and filtration through grade GF/B glass microfiber filters (Brandel Inc) using a Brandel Harvester. In 4 ml of Ecolite scintillation cocktail (MP biomedicals), [3H]ryanodine retained on the filter was counted using a Beckman LS6000 scintillation counter.
4
Measuring VSMC Proliferation and Protein Synthesis
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45,000 or 60,000 VSMCs were seeded per well in 24-well plates for [3H]-leucine and [3H]-thymidine incorporation assays, respectively. After 24 hours, cells were rendered quiescent for 48 hours, then stimulated in triplicate with positive controls or purified IgG samples (200 µg/mL). For cell proliferation assays, [3H]-thymidine (MP Biomedicals) was added to a final concentration of 0.5 µCi/mL, and for protein synthesis assays, [3H]-leucine (MP Biomedicals) was added to a final concentration of 0.3 µCi/mL. Cells were then incubated for 24 hours at 37°C, 5% CO2. After 24 hours, media was removed, and cells were washed and fixed overnight at 4°C with ice-cold 5% tri-chloro-acetic acid (Fisher). Plates were gently washed with water and once dry, the cells were solubilized in 0.1N NaOH and added to scintillation tubes containing EcoLite(+) scintillation cocktail (MP Biomedicals), and vortexed vigorously. Radioactivity was measured using a Tri-Carb 2100TR Liquid Scintillation Analyzer (Packard) as counts per minute (CPM) per well and expressed as fold change from basal.
5
Assessing Protein Interactions in Neurodegeneration
6
Quantifying Substrate Binding to H/ACA snoRNP
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Binding assays using tritium-labeled substrate RNAs were performed by incubating increasing concentrations of the substrate in the presence of 5 nM H/ACA snoRNPs reconstituted with Cbf5 wild-type or the inactive D95N variant for 3 min at 30°C. The complete 200 µL reaction was filtered through a nitrocellulose membrane, followed by washing of the nitrocellulose membrane with 1 mL cold Reaction Buffer. The nitrocellulose membrane was dissolved in 10 mL EcoLite scintillation cocktail [EcoLite (+), MP Biomedical] followed by scintillation counting to determine the amount of substrate RNA bound to the H/ACA snoRNP.
Dissociation constants (KD) were determined by fitting the binding curves to the hyperbolic function in GraphPad Prism
where [S] is the substrate concentration and Bmax is the maximum binding. The substrate RNA: Enzyme ratio was calculated by dividing the picomoles of substrate RNA retained on the nitrocellulose membrane by the picomoles of an enzyme in the reaction.
Dissociation constants (KD) were determined by fitting the binding curves to the hyperbolic function in GraphPad Prism
where [S] is the substrate concentration and Bmax is the maximum binding. The substrate RNA: Enzyme ratio was calculated by dividing the picomoles of substrate RNA retained on the nitrocellulose membrane by the picomoles of an enzyme in the reaction.
7
Ryanodine Receptor Binding Assay
8
Kinetic Analysis of tRNA Methylation by TrmB
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To determine methylation by TrmB, TrmB and [3H]SAM were rapidly mixed in a quench-flow apparatus with nonradioactive tRNAPhe to final concentrations 5 µM, 50 µM, and 1 µM, respectively, and reactions were stopped by addition of 0.1 M HCl at the same timepoints as described above. A constant volume of each quenched sample was precipitated on Whatman paper disks presoaked with 5% (w/v) trichloracetic acid. To remove unincorporated [3H]SAM, disks were washed three times with 5% (w/v) trichloracetic acid for 5 min followed by a final wash in ethanol. After drying, paper disks were added to 4 mL EcoLite(+) scintillation cocktail (MP Biomedical), and the amount of [3H]methyl incorporated was determined by scintillation counting. The apparent rate of methylation was determined by fitting to a single exponential function, as stated above. Since percent tRNA methylated cannot be determined directed with this assay, the maximum amplitude determined by fitting was set to be 100%.
9
Ryanodine Receptor Binding Assay
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In 96-well plates, HSR vesicles (1 mg/ml) and cardiac CSR vesicles (3 mg/mL) were pre-incubated with 0.02% DMSO or Hit compound, with or without 300 nM CaM, for 30 min at 22 °C in a solution containing 150 mM KCl, 5 mM GSSG, 1 µg/mL Aprotinin/Leupeptin, 1 mM EGTA, and 65 µM or 1.02 mM CaCl2 (as determined by MaxChelator to yield 30 nM or 30 μM of free Ca2+, respectively), 0.1 mg/mL BSA and 20 mM K-PIPES (pH 7.0). Non-specific and maximal [3H]ryanodine binding to SR were separately assessed by addition of 40 µM non-radioactively labeled ryanodine or 5 mM Adenylyl-imidodiphosphate, respectively. Such control samples were each distributed over 4 wells/plate. Binding of [3H]ryanodine (10 and 15 nM for cardiac and skeletal SR, respectively) was determined after a 3-h incubation (37 °C) and filtration through grade GF/B Glass Microfiber filters (Brandel Inc., Gaithersburg, MD, US) using a Brandel Harvester. In 4 mL of Ecolite Scintillation cocktail (MP biomedicals, Solon, OH, USA), [3H] on filter was counted using a Beckman LS6000 scintillation counter (Fullerton, CA).
10
Quantification of Polyphenol Standards
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