The hemiDNA 36-mer DNA duplex described above was used as substrate for measuring the steady-state methylation kinetics of DNMT1, where the Apt. #9 was used for inhibition. The 20-μL reaction contains 50 nM DNMT1, 5–400 nM hemiDNA, 0 or 0.1 μM Apt. #9, 0.5 μM S-adenosyl-l -[methyl-3H] methionine (specific activity 18 Ci/mmol, PerkinElmer), 2 μM AdoMet in 50 mM Tris–HCl, pH 8.0, 0.05% β-mercaptoethanol, 5% glycerol and 200 μg/ml BSA. The reactions were carried out in triplicate at 37°C for 0 min or 60 min, which falls into the linear range of the progression curve (Supplementary Figure S13 ), and quenched by addition of 5 μL of 10 mM AdoMet. For detection, 10 μL of reaction mixture was spot on Amersham Hybond™-XL membrane (GE Healthcare) and dried out. The membrane was then washed 3 times with 5 mL of 0.2 M cold ammonium bicarbonate (pH 8.2), 5 mL of Milli Q water and 5 mL of EtOH. Subsequently, the membrane was air dried, transferred to scintillation vials filled with 5 mL of ScintiVerse (Fisher) and subject to radioactivity measurement by a Beckman LS6500 counter. The data were analyzed by Michaelis–Menten enzymatic kinetics nonlinear regression fitting (Y = Vmax*X/(KM + X)) using GraphPad Prism7 software.
Ls6500 counter
Manufactured by Beckman Coulter
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The LS6500 is a liquid scintillation counter designed for the measurement of radioactivity in a wide range of sample types. It features advanced detection technology and intuitive software for efficient data analysis.
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Lab products found in correlation
Scintiverse, by Thermo Fisher Scientific (4 mentions)
S adenosyl l methyl 3h methionine, by PerkinElmer (3 mentions)
Biodegradable counting cocktail buffer, by Thermo Fisher Scientific (2 mentions)
S adenosyl l methyl 3h methionine adomet, by PerkinElmer (2 mentions)
Gf c filter, by GE Healthcare (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Amersham hybond xl membrane, by GE Healthcare (1 mentions)
3h ryanodine, by PerkinElmer (1 mentions)
Unlabeled ryanodine, by MP Biomedicals (1 mentions)
Electrochemiluminescence technology, by Mesoscale (1 mentions)
Dmem f12 10, by Thermo Fisher Scientific (1 mentions)
Hepes, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Hybond n nylon membrane, by GE Healthcare (1 mentions)
Deae filtermat paper, by PerkinElmer (1 mentions)
α p32 utp, by PerkinElmer (1 mentions)
15 protocols using ls6500 counter
1
Steady-State Methylation Kinetics of DNMT1
2
Protein Synthesis Measurement Protocol
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105 cells (control and Nsp2) were each plated in 12 wells of a 24-well plate with 1 ml complete D-MEM and 10% FCS (duplicate samples). The next day the medium was replaced with medium containing 5 µCi/ml L-[3,4,5-3H(N)]-Leucine (150 Ci/mmol—NEN), and sequential aliquots were removed at 1/2 h intervals starting 15 min later. After solubilization with 0.5 ml 1%SDS, 10% TCA insoluble material (0.1 mg Protein) was collected on 2.5 cm GFA filter. Following washing with 90% EtOH, the filters were air-dried and placed in scintillation vials for counting with OptiScint LLT NPE-Free Scintillation cocktail in a Beckman LS6500 counter.
3
DNMT1 DNA Methylation Assay
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DNA methylation assays were performed with 50 nM DNMT1 (aa: 351–1616) or DNMT1 (aa: 621–1616) in the presence of 1 μM 30 bp hemi-methylated dsDNA (upper strand: 5′-TTGCACTCTCCTCCXGGAAGTCCCAGCTTC-3′, X = m5C) and 1 μM S-adenosyl-l-[methyl-3H]methionine (AdoMet) (specific activity 15 Ci/mmol, PerkinElmer) in methylation buffer (20 mM HEPES, pH 7.5, 50 mM KCl, 5% glycerol, 1 mM DTT, 1 mM EDTA, 0.1 mg/ml bovine serum albumin). For comparing the effect of ubiquitin and UHRF1_UBL on DNMT1 enzymatic activity, DNMT1 proteins were pre-incubated with the indicated concentrations of ubiquitin or UHRF1_UBL on ice for 30 min. For comparing the effect of ubiquitin, histone H3 and ubiquitinated histone H3 on DNMT1 enzymatic activity, DNMT1 proteins were pre-incubated with the individual factor in methylation buffer without DTT. The reactions were carried out at 37°C and quenched at various time points from the start point to 25 min by leaving the reaction system on ice and further processed by spotting them on DE-81 membranes (GE Healthcare). The membranes were washed sequentially with 10 ml cold 0.2 M NH4HCO3, 10 ml water and 5 ml ethanol. After air-drying of the membranes, the incorporation of 3H-labeled methyl group was detected by liquid scintillation counting with a Beckman LS6500 counter.
4
Nucleotide-Binding Assay for aIF2 Protein
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The nucleotide-binding assay was performed essentially as described (20 (link)). The purified aIF2 proteins (30 picomoles) were incubated 10 min at 65°C in a buffer containing 10 mM HEPES pH7.5, 200 mM NaCl, 5 mM MgCl2, 10 mM 2-mercaptoethanol with increasing amounts (0–464 picomoles) of [3H]GDP (∼18 000 d.p.m/pmol). The final volume of the reaction was 50 μl. Aliquots (20 μl) were withdrawn and mixed with 1 ml of cold incubation buffer and then filtered through Millipore 0.22 μM nitrocellulose disks, which were washed with 2 ml of cold incubation buffer. The filters were then counted by liquid scintillation in a Beckman LS 6500 counter. Results were fitted with simple binding curves from which apparent dissociation constants and their associated standard errors were derived using the MC-Fit program (29 ).
5
Quantifying Active Ryanodine Receptors
6
Quantification of Peptide Release by RF2
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RTs at final concentrations of 0.05 or 0.02 μM (for UAA) were reacted with indicated amounts of RF2 at 37°C in a quench-flow instrument or manually, and the reactions were stopped at different time points by quenching with 17% (final concentration) formic acid. Precipitated [3H]fMet-Phe-Tyr-tRNATyr was separated from soluble [3H]fMet-Phe-Tyr peptide by 15 min centrifugation at 20 000 g and 4°C. The pellets of precipitated [3H]fMet-Phe-Tyr-tRNATyr were dissolved in 0.5 M KOH by incubating for 15 min at 37°C. The amounts of tRNA-bound and released peptides were quantified by scintillation counting of the 3H radiation in Quicksafe Flow 2 scintillation liquid in a Beckman Coulter LS 6500 counter.
Small amounts of shorter peptides, present in RT preparations, had no effect on the rate constant of peptide release (krel). 0.3 μM RTs with UAG in the A site were reacted to 3.3 μM RF2, after quenching each sample was divided into two parts and the released peptide quantified either by scintillation counting or by HPLC, as described above. The krel of total peptide release determined by scintillation counting was the same as krel of only [3H]fMet-Phe-Tyr release determined by HPLC (Supplementary Figure S2 ).
Small amounts of shorter peptides, present in RT preparations, had no effect on the rate constant of peptide release (krel). 0.3 μM RTs with UAG in the A site were reacted to 3.3 μM RF2, after quenching each sample was divided into two parts and the released peptide quantified either by scintillation counting or by HPLC, as described above. The krel of total peptide release determined by scintillation counting was the same as krel of only [3H]fMet-Phe-Tyr release determined by HPLC (
7
Metformin Release Quantification via 14C Labeling
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Carbon 14 [14C]-labeled compounds are often used to determine drug release amounts. The release of metformin was assessed following our previous method [18 (link), 19 (link)]. Briefly, 10, 30, and 50 μg of metformin (containing 1/10 [14C]-metformin) were dissolved in 15% chitosan to form the metformin + chitosan liquid. The powder and liquid portions were mixed under sterile conditions by hand spatulation. [14C] metformin-loaded scaffold was placed in 24-well plates containing 1 mL of PBS in an incubator at 37°C. At each time period, the microsphere suspension was centrifuged, and the PBS without microspheres was collected for [14C]-metformin concentration analysis. 100 μL PBS was transferred to the scintillation tube containing 3 mL Biodegradable Counting Cocktail buffer (Fisher Scientific Inc., Pittsburgh, PA). Radioactivity was counted by a multipurpose scintillation counter (Beckman LS6500 Counter, Brea, CA).
8
DNMT3A Methylation Assay Using Radiolabeled SAM
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In vitro methylation assay was performed in triplicates using 20-µL reactions containing 1 µM protein (either DNMT3AWT–DNMT3L or DNMT3AR882H–DNMT3L), 1 µM 36-mer DNA substrate with a single CG, CA, or CT site (5′-AATAATAATAATAATAACXATAATAATAATAATAAA-3′, X = G, A or T), 2.5 µM S-adenosyl-L-[methyl-3H]methionine with a specific activity of 18 Ci/mmol (PerkinElmer), 1.96 µM nonradioactive SAM, 50 mM Tris-HCl (pH 7.5), 0.05% β-mercaptoethanol, 5% glycerol and 200 µg/mL BSA. Reactions were left to proceed for 1 hour or indicated times at 37 °C and stopped by adding 5 µL of 10 mM nonradioactive SAM to each reaction. Samples were loaded onto DEAE membrane, washed three times with 0.2 M ammonium bicarbonate (pH 8.2), once with deionized water, and once with 95% ethanol. The membrane was left to dry for 1 hour, cut into squares to separate samples and soaked in a vial containing 4 mL of scintillation buffer. The incorporation of tritium onto DNA substrates was measured using Beckman LS6500 counter.
9
Characterization of Alpha TC1-6 Cell Line
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The mycoplasma-free alpha TC1–6 cell line was purchased from ATCC (CRL-2934; Barcelona), and maintained in DMEM (ThermoFisher Scientific) supplemented with 10% FBS, 15 mM HEPES, 0.1 mM non-essential amino acids, 0.02% BSA, 2 g/l glucose, 1.5 g/l sodium bicarbonate and 5 µM beta mercaptoethanol (all purchased from Sigma-Aldrich). The proliferation of splenocytes isolated from mouse spleens was assessed after a 3-day culture in RPMI 1640 medium supplemented with 8% FBS, 20 mM l -glutamine, 1% sodium pyruvate, 1% nonessential amino acids, and 1% penicillin/streptomycin (all from Invitrogen), in the presence or absence of the insulin peptide SLYQLENYCA. Cells were pulsed with [3H]-thymidine for the last 24 h of culture, harvested and lysed onto membranes prior to liquid scintillation counting using a Beckman Coulter LS 6500 counter. Mouse primary macrophages were isolated from the peritoneal cavity, and cultured in DMEM/F12–10 (ThermoFisher Scientific) supplemented with 10% FBS, 2 mM l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (all purchased from Sigma-Aldrich). Cells were stimulated with 1 μg/ml LPS (in DMSO), in the absence or presence of 0.1, 1, or 10 μM BL001 for 24 h. The secretion of cytokines was measured in the culture medium by electrochemiluminescence technology from MesoScale Discovery (Rockville, USA), and RNA was extracted from cells.
10
DNA Methylation Assay for CG, CA, and CT Substrates
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