In vitro methylation reactions were carried out in 30 μl of phosphate-buffered saline (pH = 7.4.) containing 0.5–1.0 μg of substrate, 3 μg of recombinant enzymes and 0.42 μM S-adenosyl-l-[methyl-3H]methionine (79 Ci/mmol from a 7.5 μM stock solution; PerkinElmer Life Sciences). The reaction was incubated at 30° C for 1 h and then separated on SDS-PAGE, transferred to a PVDF membrane, treated with En3Hance™ (PerkinElmer Life Sciences), and exposed to film for 1–3 days at −80° C.
S adenosyl l methyl 3h methionine
Manufactured by PerkinElmer
Sourced in United States
S-adenosyl-L-[methyl-3H] methionine is a radioactive compound used as a tracer in various biochemical and biological research applications. It serves as a methyl group donor in methylation reactions, which are important cellular processes. This product is intended for research use only and should be handled with appropriate safety precautions.
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Lab products found in correlation
En3hance, by PerkinElmer (12 mentions)
Memcode reversible stain, by Thermo Fisher Scientific (4 mentions)
Ls6500 counter, by Beckman Coulter (3 mentions)
Scintiverse, by Thermo Fisher Scientific (2 mentions)
Hybond, by Cytiva (2 mentions)
S adenosyl l methionine, by Merck Group (2 mentions)
A7007, by Merck Group (2 mentions)
Whatman, by Cytiva (2 mentions)
Amersham hybond xl membrane, by GE Healthcare (1 mentions)
Tlc plate, by Merck Group (1 mentions)
Any kd mini protean tgx precast gel, by Bio-Rad (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
Memcode reversible protein stain, by Thermo Fisher Scientific (1 mentions)
En3hance spray surface autoradiography enhancer, by PerkinElmer (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Prism 6, by GraphPad (1 mentions)
Ultima gold scintillation cocktail, by PerkinElmer (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Ls 6500 multi purpose scintillation counter, by Beckman Coulter (1 mentions)
33 protocols using s adenosyl l methyl 3h methionine
1
In vitro Methylation Assay
2
In vitro Methyltransferase Assay Procedure
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In vitro methyltransferase assays were performed as described previously [47 (link)–49 (link)]. Briefly, purified His-SUV39H2 expressed in Baculovirus infected insect cells or purified His-SUV39H2 with purified histone H3 expressed in E.coli or purified LSD1 expressed in Baculovirus infected insect cells was incubated in 50 mM Tris-HCl (pH 8.8) buffer with 1.0 μCi/ml S-adenosyl-L-[methyl-3H]-methionine (Perkin Elmer, Waltham, MA) for 2 hours at 30°C. After boiling in sample buffer, the samples were subjected to SDS-PAGE, and visualized by autoradiography [50 (link)].
3
Steady-State Methylation Kinetics of DNMT1
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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.
4
In vitro PRMT Methylation Assay
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In vitro methylation reactions were performed in 30 μl PBS (pH 7.4) with 1 μg of peptide, 1 μg of recombinant enzymes (PRMT1, CARM1 or PRMT9) and 0.42 μM S-adenosyl-l-[methyl-3H]methionine (Perkin Elmer). Reactions were incubated at 30 °C for 1 hour, resolved on 15% SDS-PAGE, transferred to PVDF membrane, treated with En3Hance (Perkin Elmer) and exposed to film at −80 °C. Exposure time was different for each PRMT: PRMT1 for 3 days, CARM1 for 2 days, and PRMT9 for 12 days.
5
Protein Methyltransferase Activity Assay
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We incubated 1.4 μg of purified recombinant PhoP proteins at 30°C overnight with 2.8 μg of recombinant putative protein methyltransferases in 20 μl of methylation buffer (20 mM Tris-HCl, 150 mM NaCl, and 1 mM EDTA, pH 7.5) supplemented with 1 μCi of S-adenosyl-l -[methyl-3H] methionine (PerkinElmer) or 0.2 mM cold SAM (NEB). Reactions were stopped by the addition of 2×SDS-PAGE sample buffer and heating. Samples were separated in 12% SDS-PAGE and followed by Western blotting or autoradiography. For autoradiography, the gels were fully dry by gel dryer (Wadali) and then exposed to X-ray film.
6
Radioactive Assay for Juvenile Hormone Biosynthesis
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S-Adenosyl-L-methionine (SAM) was purchased from Sigma-Aldrich and S-Adenosyl-L-[methyl-3H] methionine (370GBq mmol, 10 Ci/mmol) from Perkin-Elmer Life Sciences (Waltham). Methyltransferase activity in the brain-RG complexes isolated from larvae at 3hAIW was measured with JHA and FA as substrates, as described previously [14 (link), 20 (link), 23 (link), 33 ]. L-[Metyl-3H] methionine (2.92–3.70 TBq/mmol) was purchased from Perkin-Elmer Life Sciences and TLC plates (20×20 cm2 plastic plate coated with silica gel F254) from Merck KgaA (Germany). JH biosynthesis in the brain-RG complexes was detected using the radiochemical assay followed by thin layer chromatography analysis as reported previously [18 (link), 19 (link), 37 (link)]. JH titers from the whole bodies of each genotype were determined using the recently developed HPLC-FD protocol [34 (link)].
7
In vitro Protein Methylation Assay
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In vitro methylation reactions were carried out in 30 μl of phosphate-buffered saline (pH = 7.4.) containing 0.5–1.0 μg of substrate, 3 μg of recombinant enzymes and 0.42 μM S-adenosyl-l- [methyl-3H]methionine (79 Ci/mmol from a 7.5 μM stock solution; PerkinElmer Life Sciences). The reaction was incubated at 30°C for 1 h and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a Polyvinylidene difluoride (PVDF) membrane, treated with En3Hance™ (PerkinElmer Life Sciences) and exposed to film for 1 day at −80°C. After exposure, the PVDF membrane was washed with methanol and stained with coomassie to visualize total protein loaded.
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In vitro methylation reactions were carried out in 30 μl of phosphate-buffered saline (pH = 7.4.) containing 0.5–1.0 μg of substrate, 3 μg of recombinant enzymes and 0.42 μM S-adenosyl-l- [methyl-3H]methionine (79 Ci/mmol from a 7.5 μM stock solution; PerkinElmer Life Sciences). The reaction was incubated at 30°C for 1 h and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a Polyvinylidene difluoride (PVDF) membrane, treated with En3Hance™ (PerkinElmer Life Sciences) and exposed to film for 1 day at −80°C. After exposure, the PVDF membrane was washed with methanol and stained with coomassie to visualize total protein loaded.
8
Methylation Assay for PTEN by SMYD2
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In vitro methyltransferase assays were described previously [17,19–22] . Briefly, recombinant PTEN protein was incubated with recombinant SMYD2 and 2 μCi S-adenosyl-l -[methyl-3H]-methionine (PerkinElmer, Waltham, MA) in a mixture of methylase activity buffer (50 mM Tris-HCl at pH 8.8, 10 mM DTT, and 10 mM MgCl2) for 1 hour at 30°C. After denaturing, samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), blotted to polyvinylidene difluoride membrane, and visualized by MemCode Reversible Stain (Thermo Fisher Scientific, Waltham, MA) and fluorography.
9
In Vitro Methylation Assay
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In vitro methylation reactions were carried out in 30 μl of phosphate-buffered saline (PBS) (pH = 7.4.) containing 0.5–1.0 μg of substrate, 3 μg of recombinant enzymes, and 0.42 μM S-adenosyl-l-[methyl-3H] methionine (79 Ci/mmol from a 7.5 μM stock solution; PerkinElmer Life Sciences). The reactions were incubated at 30 °C for 1 h and then separated on SDS-PAGE, transferred to a PVDF membrane, treated with En3Hance™ (PerkinElmer Life Sciences), and exposed to film for 1–3 days at –80 °C.
In vitro methylation with total cell lysates as substrates was performed as described above except that total cell lysates from HeLa cells, mESCs, E16 mouse embryo, and various mouse tissues were used as the substrates. Tissue and cell lysates were prepared with a mild lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% NP-40, 15 mM MgCl2). The lysate was sonicated to maximize the protein extraction.
In vitro methylation with total cell lysates as substrates was performed as described above except that total cell lysates from HeLa cells, mESCs, E16 mouse embryo, and various mouse tissues were used as the substrates. Tissue and cell lysates were prepared with a mild lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% NP-40, 15 mM MgCl2). The lysate was sonicated to maximize the protein extraction.
10
In vitro Methylation Assay
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The recombinant proteins of GST-USP9X C2, GST-PRMT1, GST-PRMT3 and GST-CARM1 were purified from bacterial. In vitro methylation reactions were carried out in 30 μl of phosphate-buffered saline (pH=7.4.) containing 0.5–1.0 μg of substrate, 3 μg of recombinant enzymes and 0.42 μm S-adenosyl-l-[methyl-3H]methionine (79 Ci/mmol from a 7.5 μm stock solution; PerkinElmer Life Sciences, Boston, MA, USA). The reaction was incubated at 30 °C for 1 h and then separated on SDS–PAGE, transferred to a PVDF membrane, treated with En3Hance (PerkinElmer Life Sciences), and exposed to film for 2 weeks at −80 °C.
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