The peptide arrays were pre‐incubated in protein specific methylation buffer (HEMK2/TRMT112: 10 mM Tris/HCl pH 7.6, 50 mM KCl, 10 mM Mg/OAc, 1 mM DTT; NSD1/NSD2/SMYD2/SUV39H1/SUV39H2: 50 mM Tris/HCl pH 9, 5 mM MgCl2, 4 mM DTT; SET7/9: 20 mM Tris/HCl pH 9, 5 mM DTT; SETD6: 50 mM Tris/HCl pH 9, 5 mM DTT; SET8: 20 mM HEPES pH 8, 50 mM NaCl, 5 mM DTT) on a shaker for 5 min at room temperature. After this pre‐incubation step, the peptide arrays were methylated in methylation buffer containing the different PKMTs at concentrations mentioned in the text in the presence of 0.76 μM labeled [methyl‐3H]‐AdoMet (Perkin Elmer Inc., dissolved at 25 μM in 10 mM sulfuric acid) at room temperature. Afterwards, the peptide arrays were washed 5 times for 5 min each in 100 mM NH4HCO3 and 1% SDS and 5 min in Amplify NAMP 100 V from GE Healthcare. Once complete, arrays were exposed to Hyperfilm™ high‐performance autoradiography films (GE Healthcare) in the dark at −80°C. Film development was performed with an Optimus TR developing machine.
Methyl 3h adomet
Manufactured by PerkinElmer
Sourced in United States
[methyl‐3H]‐AdoMet is a radiolabeled form of S‐adenosylmethionine (AdoMet), a naturally occurring co‐factor involved in various methylation reactions. It contains a tritium (3H) label at the methyl group, making it suitable for use in studies of methyltransferase enzyme activity and related biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Amplify namp100v, by GE Healthcare (3 mentions)
Scintillation counting, by Beckman Coulter (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Hyperfilm high performance autoradiography film, by GE Healthcare (2 mentions)
Rna probe, by Thermo Fisher Scientific (2 mentions)
Anti h4, by Merck Group (1 mentions)
Amersham hyperfilm mp, by Cytiva (1 mentions)
En3hance solution, by PerkinElmer (1 mentions)
Origin 7, by OriginLab (1 mentions)
De81 paper circles, by Cytiva (1 mentions)
Amplify solution, by GE Healthcare (1 mentions)
Ni sepharose 6 fast flow, by GE Healthcare (1 mentions)
14 protocols using methyl 3h adomet
1
Methylation Assay for Histone Methyltransferases
2
In vivo Methyl Group Labeling
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Overnight cultures of WT, hpm1Δ, and rpl3-H243A cells were diluted in fresh YPD media to an OD600 nm of 0.1 and grown at 30°C in a rotary shaker until the OD600 nm reached 0.8–1.0. Seven OD600 nm units from each culture were harvested by centrifugation at 5000g for 5 min at room temperature and the cells were washed three times with water. Cells were then resuspended in 848 µL of YPD and 152 µL of S-adenosyl-[methyl-3H]-l -methionine ([methyl-3H]AdoMet; PerkinElmer; 83.3 Ci/mmol; 0.55 mCi/mL in 10 mM H2SO4:ethanol 9:1) and incubated at 30°C in a rotary shaker for 30 min. Cells were then harvested by centrifugation and washed several times with water.
3
Methylation Analysis of Histone and ERF1
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His‐tagged ERF1‐WT/Q185K and GST‐tagged H4 WT/K12Q proteins or recombinant nucleosomes were incubated with HEMK2/TRMT112 or SETD6 in methylation buffer supplemented with 0.76 μM labeled [methyl‐3H]‐AdoMet (Perkin Elmer) at 25°C. Concentrations and methylation times are stated in the text. The methylation reactions were stopped by the addition of SDS loading buffer and boiling for 5 min at 95°C. Equal loading of target protein amounts was confirmed by Coomassie Brilliant Blue staining and Western Blot using as primary antibody anti‐H4 (Millipore, 05‐858, Lot: 3768156) for GST‐tagged H4 or anti‐His (Qiagen, Hilden, Germany) for His‐tagged ERF1. The methylated protein samples were separated by 16% SDS‐PAGE, whereas methylated nucleosome samples were separated by tricine gel electrophoresis. Then, the gels were incubated for 1 h in Amplify NAMP 100 V (GE Healthcare) and dried for 90 min at 70°C under vacuum. The dried gels were exposed to Hyperfilm™ high‐performance autoradiography films (GE Healthcare) in the dark at −80°C. Film development was performed with an Optimus TR developing machine.
4
AdoMet Cross-linking of Purified Enzyme
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AdoMet cross-linking was performed as described8 (link). An aliquot of 2 µg of purified enzyme with 0.5 µCi of [methyl-3H]AdoMet (17.9 Ci/mmol, PerkinElmer) in 20 µl of 50 mM BisTris propane (pH 8.5), 1 mM EDTA, and 0.5 mM DTT, were transferred to a 96-well plate on ice and placed 8 cm from an inverted UV transilluminator (Compact UV Lamp, UVGL-25 (Analytik Jena US)) using UV-C (254 nm wavelength) for 1 h. The protein was then separated by SDS-PAGE, stained with Coomassie, and subjected to fluorography. The gels were incubated in water for 30 min, then incubated in 5× volume of EN3HANCE solution (Perkin Elmer) for 1 h. Gels were washed in 1% glycerol for 1 h and dried. An X-ray film (Amersham Hyperfilm MP (Cytiva)) was exposed to the dried gel for 48 h at −80 °C, followed by development of the X-ray film.
5
Kinetic Analysis of Recombinant Enzymes
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Radioactive methylation assays to determine kinetic parameters of recombinant enzymes were performed using 3H-labeled S-adenosylmethionine (AdoMet) as a methyl group donor and biotinylated oligonucleotides of varying sizes bound on avidin-coated high-binding Elisa plates (Corning) as described (43 (link)). The DNA methylation reactions were carried out using either 250 nM 30-bp/32-bp DNA substrate in methylation buffer (20 mM HEPES pH 7.5, 50 mM KCl and 1 mM EDTA, 5 μg/ml BSA). The methylation reaction included 0.76 μM [methyl3H] AdoMet (PerkinElmer Life Sciences). Storage buffer was added to compensate for the different enzyme volumes in all reactions. Incorporated radioactivity was quantified by scintillation counting.
6
Hsp70 RNA Methylation Assay
7
Peptide Methylation Assay with MLL1-SET
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For peptide methylation, a microplate assay was used basically as described (Gowher et al., 2005 ). MLL1‐SET (0.8 μm ) was incubated in the absence or presence of equimolar amounts of complex proteins with 0.625 μm biotinylated H3 (1–19) peptide (Intavis) in methylation buffer (50 mm Tris/HCl pH 8, 200 mm NaCl, 5 mm MgCl2, and 3 mm DTT) containing 0.76 μm radioactively labeled [methyl‐3H]‐AdoMet (PerkinElmer Life Sciences, Boston, MA, USA) for 3 h at 22 °C in an Eppendorf tube. Afterward, the samples were transferred to an avidin‐coated microplate (Greiner, Bio‐One, Frickenhausen, Germany) and shaken for 30 min. To remove unbound peptide, the microplate was washed with 1× PBST and 500 mm NaCl. For elution of the bound peptide, 50 mm HCl was added and incubated for 1 h. The released radioactivity was analyzed by liquid scintillation counting in a Hidex 300SL (HIDEX, Mainz, Germany).
8
DNMT1 Enzymatic Activity Assay
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The enzymatic activities of DNMT1 proteins were assessed by incorporation of 3H-labelled methyl group from S-adenosyl-l-[methyl-3H]methionine ([methyl-3H]AdoMet, PerkinElmer). The substrate used in this assay was 14-bp hemi-methylated dsDNA (5′- GGAGGCXGCCTGCT -3′; in the top strand, X=5 mC, whereas in the bottom strand X=C, with a 5′-biotin label for purification). DNMT1 (0.3 μM) and USP7 (3 μM) proteins were mixed and incubated on ice for 15 min. The biotinylated DNA (2 μM) was methylated by DNMT1 proteins in the presence of 2.5 μM [methyl-3H]AdoMet, 25 mM Tris-HCl (pH 7.5) and 1 mg ml−1 BSA. The reactions were incubated at 37 °C for 30 min and were terminated with the addition of cold wash buffer (500 mM NaCl and 1 mM EDTA in PBST). The DNA products were immobilized on streptavidin beads, washed three times and subjected to liquid-scintillation counting (PerkinElmer). Each reaction was performed in triplicate.
9
In Vitro RNA Methylation Assay
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ER:HRasG12V cells with or without 48-hour 4-OHT treatment (1 μM) were lysed in denaturing lysis buffer [20 mM tris-HCl (pH 7.5), 137 mM NaCl, 1% NP-40, and 2 mM EDTA] after three times wash with cold DPBS, then incubated at 4°C for 30 min with rotation, followed by centrifuging at 12,000 rpm for 15 min. The supernatant were collected and the concentration of total protein was measured using 660-nm protein assay reagent (Pierce). The in vitro methylation assay was performed in a 50-μl reaction mixture containing 400 nM RNA probe (commercially synthesized in vitro, Thermo Fisher Scientific), cell lysate with same amount of total protein, 20 mM tris (pH 7.5), 50 μM ZnCl2, 1 mM DTT, RNaseOUT (0.2 U/μl), 1% glycerol, and 0.5 μCi [methyl-3H]AdoMet (PerkinElmer). The reaction was incubated at 30°C for 1 hour and then stopped by adding TRIzol reagent (Invitrogen). RNA after reaction was precipitated and purified using sodium acetate at −20°C for at least 2 hours. The precipitated RNA was subjected to radioactivity measurement using scintillation counting (Beckman). Levels of 3H-methyl–incorporated RNA are shown as disintegrations per minute.
10
DNMT1 Methyltransferase Inhibition Assay
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For DNMT1, methyl transfer activity
inhibition assays were performed in 20 μL reactions containing
4.6 mM [methyl-3H]-AdoMet (10.0 Ci/mmol; PerkinElmer),
1.0 mM DNA oligonucleotides, 0.2 μM DNMT1, 1 mM EDTA, and 50
mM Tris-HCl, pH 7.5. The DNA substrates were 36 bp hemimethylated
(GAC)12. Enzymes were preincubated with AdoMet and various
concentrations of inhibitors for 5 min at 37 °C before the addition
of substrate DNA. After a 15 min incubation, the reactions were terminated
by the addition of 1% SDS and 1 mg/mL of protease K and heating at
50 °C for 15 min. The reaction mixtures were spotted on DE81
paper circles (Whatman), washed with 5 mL of cold 0.2 M NH4HCO3 (twice), 5 mL of deionized water (twice), and 5 mL
of ethanol (once). The dried circles were subjected to liquid scintillation
counting with Cytoscint scintillant. All curves were fit individually
using Origin 7.5 software (OriginLab).
inhibition assays were performed in 20 μL reactions containing
4.6 mM [methyl-3H]-AdoMet (10.0 Ci/mmol; PerkinElmer),
1.0 mM DNA oligonucleotides, 0.2 μM DNMT1, 1 mM EDTA, and 50
mM Tris-HCl, pH 7.5. The DNA substrates were 36 bp hemimethylated
(GAC)12. Enzymes were preincubated with AdoMet and various
concentrations of inhibitors for 5 min at 37 °C before the addition
of substrate DNA. After a 15 min incubation, the reactions were terminated
by the addition of 1% SDS and 1 mg/mL of protease K and heating at
50 °C for 15 min. The reaction mixtures were spotted on DE81
paper circles (Whatman), washed with 5 mL of cold 0.2 M NH4HCO3 (twice), 5 mL of deionized water (twice), and 5 mL
of ethanol (once). The dried circles were subjected to liquid scintillation
counting with Cytoscint scintillant. All curves were fit individually
using Origin 7.5 software (OriginLab).
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