The methyltransferase assay was carried out at 30°C for one hour in a total volume of 25 μl containing 1.5 μl of S-adenosyl-l-[methyl-3H] methionine (SAM) (14.4 Ci/mmol; PerkinElmer), 1.5 μl substrate DNA (12 repeats of TAC, annealed to form dsDNA, 15 μM), and 0.2 μM AtDRM2 full length (59–626) or DRM2 methyltransferase (DRM2CAT, 269–626) proteins, 1 μM His-tag UVR8 or GFP proteins in assay buffer (20 mM MOPS [pH 7.0], 1 mM DTT, 5 mM EDTA, 200 μg/ml BSA, and 5% glycerol). The reactions were stopped by adding 1 μl of cold SAM (NEB). A total of 11 μl from each reaction was applied onto DEAE Filtermat (PerkinElmer,1450–522) and washed two times with 200 mM ammonium bicarbonate, two times with water, and two times with ethanol. The paper was dried and placed into 4 mL of liquid scintillation cocktail (Fisher Scientific) and the activity was measured by Liquid Scintillation Analyzer (PerkinElmer, Tri-Carb 2910 TR).
S adenosyl l methyl 3h methionine sam
Manufactured by PerkinElmer
S-adenosyl-l-[methyl-3H] methionine (SAM) is a radioactively labeled compound used in research applications. It is a metabolite involved in methylation reactions.
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4 protocols using s adenosyl l methyl 3h methionine sam
1
Methyltransferase Assay for DNA Methylation
2
In vitro Methyltransferase Assay Protocol
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In vitro methyltransferase assays were described previously [37 (link), 39 (link)–46 (link)]. Briefly, recombinant GST-tagged INCENP (amino acids 821-918) proteins were incubated with recombinant PRMT1 and 2 μCi S-adenosyl-L-[methyl-3H]-methionine (SAM) (Perkin Elmer, 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 2 h at 30°C. After denaturing, samples were separated by SDS-PAGE, blotted to PVDF membrane and visualized by MemCodeTM Reversible Stain (Thermo Fisher Scientific, Waltham, MA) and fluorography.
3
Quantitative DNA Methylation Assay
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The DNA methylation assays were performed as previously described29 (link). In essence, each reaction mixture contains 0.1 μM hDNMT1351–1600, wild type or mutants, 0.5 μM S-adenosyl-l -[methyl-3H] methionine (SAM) (Perkin Elmer), 0.4 μM (GTmC)12/(GAC)12 hemimethylated DNA duplex, or various amount of histone peptides in 50 mM Tris-HCl (pH 8.0), 7 mM β-ME, 5% glycerol, 100 μg/mL BSA, and 100 mM NaCl. Each reaction lasted 20 min at 37 °C, before being quenched by 2 mM cold SAM. The activity was measured by Beckman LS6500 scintillation counter.
4
Methyltransferase Assay for DNA Methylation
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The methyltransferase assay was carried out at 30°C for one hour in a total volume of 25 μl containing 1.5 μl of S-adenosyl-l-[methyl-3H] methionine (SAM) (14.4 Ci/mmol; PerkinElmer), 1.5 μl substrate DNA (12 repeats of TAC, annealed to form dsDNA, 15 μM), and 0.2 μM AtDRM2 full length (59–626) or DRM2 methyltransferase (DRM2CAT, 269–626) proteins, 1 μM His-tag UVR8 or GFP proteins in assay buffer (20 mM MOPS [pH 7.0], 1 mM DTT, 5 mM EDTA, 200 μg/ml BSA, and 5% glycerol). The reactions were stopped by adding 1 μl of cold SAM (NEB). A total of 11 μl from each reaction was applied onto DEAE Filtermat (PerkinElmer,1450–522) and washed two times with 200 mM ammonium bicarbonate, two times with water, and two times with ethanol. The paper was dried and placed into 4 mL of liquid scintillation cocktail (Fisher Scientific) and the activity was measured by Liquid Scintillation Analyzer (PerkinElmer, Tri-Carb 2910 TR).
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