s adenosylmethionine  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    S adenosylmethionine SAM 32mM
    Description:
    S adenosylmethionine SAM 32mM 0 5 ml
    Catalog Number:
    B9003S
    Price:
    38
    Size:
    0 5 ml
    Category:
    Biochemical Reagents
    Score:
    85
    Buy from Supplier


    Structured Review

    New England Biolabs s adenosylmethionine
    S adenosylmethionine SAM 32mM
    S adenosylmethionine SAM 32mM 0 5 ml
    https://www.bioz.com/result/s adenosylmethionine/product/New England Biolabs
    Average 99 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    s adenosylmethionine - by Bioz Stars, 2019-10
    99/100 stars

    Images

    1) Product Images from "Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori"

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori

    Journal:

    doi: 10.1128/JB.00108-07

    Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.
    Figure Legend Snippet: Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.

    Techniques Used: Purification, Incubation, Polymerase Chain Reaction, Concentration Assay, In Vitro

    2) Product Images from "Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF"

    Article Title: Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp524

    ( A ) Native electrophoresis of SNF2H remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of SNF2H (lane 2: 6 nM; lane 3: 19 nM; lanes 4 and 5: 57 nM) in the presence (lanes 2–4) or absence (lane 5) of ATP (1 mM) as indicated on top, for 1 h at 30°C. Reactions were stopped by addition of ADP (10 mM) and incubation on ice for 10 min. Methylation of the remodeled products were performed as follow. The reactions were incubated for 15 min at 37°C after addition of M.SssI (5 U) and S-adenosylmethionine (160 μM). The remodeling reactions were separated by native gel electrophoresis after addition of competitor plasmid DNA and visualized by ethidium bromide staining. Gel areas excised and used for analysis are delimited by a black frame. Back frames are connected by dashed lines when gel slices were combined. ( B–F ) Schematic representation of individual DNA molecules remodeled by SNF2H. Bisulfite-converted DNAs from gel slices (black frames, lanes 3–5) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 2 D. The number of remodeled molecules shown is proportional to the average intensity of the bands generated after remodeling at enzyme concentration allowing maximal remodeling in three independent experiments. ( G ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel F (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–E (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.
    Figure Legend Snippet: ( A ) Native electrophoresis of SNF2H remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of SNF2H (lane 2: 6 nM; lane 3: 19 nM; lanes 4 and 5: 57 nM) in the presence (lanes 2–4) or absence (lane 5) of ATP (1 mM) as indicated on top, for 1 h at 30°C. Reactions were stopped by addition of ADP (10 mM) and incubation on ice for 10 min. Methylation of the remodeled products were performed as follow. The reactions were incubated for 15 min at 37°C after addition of M.SssI (5 U) and S-adenosylmethionine (160 μM). The remodeling reactions were separated by native gel electrophoresis after addition of competitor plasmid DNA and visualized by ethidium bromide staining. Gel areas excised and used for analysis are delimited by a black frame. Back frames are connected by dashed lines when gel slices were combined. ( B–F ) Schematic representation of individual DNA molecules remodeled by SNF2H. Bisulfite-converted DNAs from gel slices (black frames, lanes 3–5) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 2 D. The number of remodeled molecules shown is proportional to the average intensity of the bands generated after remodeling at enzyme concentration allowing maximal remodeling in three independent experiments. ( G ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel F (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–E (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

    Techniques Used: Electrophoresis, Incubation, Methylation, Nucleic Acid Electrophoresis, Plasmid Preparation, Staining, Amplification, Polymerase Chain Reaction, Clone Assay, Generated, Concentration Assay

    Related Articles

    Methylation Sequencing:

    Article Title: MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma
    Article Snippet: Methylation analyses of STK26 CpG island promoter region were carried out using co mbined b isulfite and r estriction a nalyses (CoBRA) and direct bisulfite sequencing. .. Prior to bisulfite conversion, fully methylated positive controls were also generated by incubating samples with S-Adenosyl methionine (SAM) and DNA methyltransferase (New England Biolabs) at 37°C for 2 hr.

    Clone Assay:

    Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation
    Article Snippet: Paragraph title: Cloning and in Vitro Methylation of pCpG Free-L1 Vector ... Briefly, 1 μg of plasmid DNA was added to a reaction containing CpG methyltransferase (Sss1, 4 units/μl) in the presence of 160 μ m S -adenosylmethionine (New England Biolabs) and incubated for 4 h at 37 °C, and S -adenosylmethionine was replenished after every 2 h. Unmethylated control reaction contained the NPC1L1 promoter construct and methylases but not S -adenosylmethionine.

    Article Title: Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells
    Article Snippet: Paragraph title: Cloning of the upstream region of precursor mir-142 , construction of expression constructs, in vitro DNA methylation and luciferase experiments ... 18 µg of pCpGL/2031 were in vitro methylated using the CpG methyltransferase M.SssI (4 units/µg), in the presence of 640 µM S-Adenosylmethionine (both from New England Biolabs) for 3 hours at 37°C and designated pCpGL/2031_M.SssI.

    Amplification:

    Article Title: Curcumin Down-Regulates DNA Methyltransferase 1 and Plays an Anti-Leukemic Role in Acute Myeloid Leukemia
    Article Snippet: The primers for amplification of p15INK4B and its bisulfite-converted promoter region, and also for DNMT1 and its Sp1-binding promoter region, were purchased from Sigma-Aldrich or Integrated DNA Technology (IDT, Coralville, IA). .. M. SssI methylase, s-adenosylmethionine (SAM) (3.2 mM) and 10X incubation buffer were purchased from New England Biolabs (Ipswich, MA).

    Article Title: Dietary Flavones as Dual Inhibitors of DNA Methyltransferases and Histone Methyltransferases
    Article Snippet: The substrate DNA for an in vitro methylation assay was a 721 bp fragment (-428/+243 relative to the initiation codon), which was amplified from RWPE-1 cells within the promoter region of the human GSTP1 gene. .. The methylation reactions were carried out in 1X M.SssI buffer with 160 mM SAM S-adenosylmethionine (supplied with M.SssI by New England Biolabs).

    Article Title: SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC
    Article Snippet: Hemimethylated linear templates were generated from fully methylated PCR products by performing one additional PCR cycle. oriC was first amplified with 22 bp PCR primers annealing at position −184 and 343 (numbered as in ), to generated a 527 bp fragment. .. PCR products (2μg) purified using a QIAquick PCR purification kit were treated with 8 units of dam methylase and 800μM S-adenosylmethionine (New England Biolabs) at 37°C overnight.

    Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation
    Article Snippet: For the construction of pCpG free L1 vector, the DNA fragment containing −1741/+56 region of the human NPC1L1 promoter was amplified using forward 5-ATCGATGCGAGTACTTGGACTCTATCTCTCTGTGG-3′ and reverse 5-ATCGATGCGAAGCTTCCCAGGTCTGGGAAGGGGTCA-3′ primers, and the amplified promoter fragments were then inserted into a promoterless CpG free vector (pCpG free basic lucia, InvivoGen, San Diego) upstream of the lucia reporter gene. .. Briefly, 1 μg of plasmid DNA was added to a reaction containing CpG methyltransferase (Sss1, 4 units/μl) in the presence of 160 μ m S -adenosylmethionine (New England Biolabs) and incubated for 4 h at 37 °C, and S -adenosylmethionine was replenished after every 2 h. Unmethylated control reaction contained the NPC1L1 promoter construct and methylases but not S -adenosylmethionine.

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori
    Article Snippet: The results were evaluated using a PhosphorImager (Molecular Dynamics Inc.). .. To investigate methylation and protection by the recombinant protein, we incubated 1 μg of a PCR fragment containing one GANTC site (778 bp, amplified with 1351GANTCF and 1351GANTCR) (Table ) with NEB2 buffer, S -adenosylmethionine (New England BioLabs), and different M.HpyAIV concentrations (0, 200, 400, 800, and 1,200 nM) and performed incubation for 1 h at room temperature followed by protein inactivation at 95°C for 10 min. .. Samples were digested with HinfI overnight at 37°C.

    Article Title: Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells
    Article Snippet: The 2,031 bp upstream region of pre-mir-142 was amplified using PCR and The AccuPrime Taq DNA Polymerase High Fidelity (Life Technologies) ( ) and T/A-cloned into the CpG-free pCpGL-basic luciferase reporter construct, which was a gift from Michael Rehli . .. 18 µg of pCpGL/2031 were in vitro methylated using the CpG methyltransferase M.SssI (4 units/µg), in the presence of 640 µM S-Adenosylmethionine (both from New England Biolabs) for 3 hours at 37°C and designated pCpGL/2031_M.SssI.

    Article Title: CREB/ATF-dependent T cell receptor-induced FoxP3 gene expression: a role for DNA methylation
    Article Snippet: The PCR product was cloned using the TOPO TA Cloning kit (Invitrogen). .. For plasmid methylation, the +4,301 to +4,500 CpG island region was amplified by PCR, and 5 μg of PCR product was incubated with 80 U of SssI methylase and 640 μM S-adenosylmethionine (New England Biolabs) for 2 h at 37°C. .. DNA methylation was verified using AatII, which is a methylation-sensitive restriction enzyme.

    Synthesized:

    Article Title: Structure of the Arginine Methyltransferase PRMT5-MEP50 Reveals a Mechanism for Substrate Specificity
    Article Snippet: For activity assays 10 µg/mL PRMT5-MEP50 complex was incubated in KCl/HEPES buffer (100 mM KCl (pH 7.5), 20 mM HEPES, 1 mM EDTA, 0.1 mM DTT, and 10% glycerol) in presence or absence of 1.5 mM S-adenosylmethionine (New England Biolabs, Ipswich, MA). .. For activity assays 10 µg/mL PRMT5-MEP50 complex was incubated in KCl/HEPES buffer (100 mM KCl (pH 7.5), 20 mM HEPES, 1 mM EDTA, 0.1 mM DTT, and 10% glycerol) in presence or absence of 1.5 mM S-adenosylmethionine (New England Biolabs, Ipswich, MA).

    Construct:

    Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation
    Article Snippet: Cloned vector was then methylated in vitro utilizing Ss1 methylases (New England Biolabs, Frankfurt, Germany) according to the manufacturer's instruction. .. Briefly, 1 μg of plasmid DNA was added to a reaction containing CpG methyltransferase (Sss1, 4 units/μl) in the presence of 160 μ m S -adenosylmethionine (New England Biolabs) and incubated for 4 h at 37 °C, and S -adenosylmethionine was replenished after every 2 h. Unmethylated control reaction contained the NPC1L1 promoter construct and methylases but not S -adenosylmethionine. .. Plasmid DNA was then purified by using Qiagen miniprep kit and quantified using a spectrophotometer.

    Article Title: Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells
    Article Snippet: Paragraph title: Cloning of the upstream region of precursor mir-142 , construction of expression constructs, in vitro DNA methylation and luciferase experiments ... 18 µg of pCpGL/2031 were in vitro methylated using the CpG methyltransferase M.SssI (4 units/µg), in the presence of 640 µM S-Adenosylmethionine (both from New England Biolabs) for 3 hours at 37°C and designated pCpGL/2031_M.SssI.

    Microarray:

    Article Title: Structure of the Arginine Methyltransferase PRMT5-MEP50 Reveals a Mechanism for Substrate Specificity
    Article Snippet: For activity assays 10 µg/mL PRMT5-MEP50 complex was incubated in KCl/HEPES buffer (100 mM KCl (pH 7.5), 20 mM HEPES, 1 mM EDTA, 0.1 mM DTT, and 10% glycerol) in presence or absence of 1.5 mM S-adenosylmethionine (New England Biolabs, Ipswich, MA). .. For activity assays 10 µg/mL PRMT5-MEP50 complex was incubated in KCl/HEPES buffer (100 mM KCl (pH 7.5), 20 mM HEPES, 1 mM EDTA, 0.1 mM DTT, and 10% glycerol) in presence or absence of 1.5 mM S-adenosylmethionine (New England Biolabs, Ipswich, MA).

    Incubation:

    Article Title: Curcumin Down-Regulates DNA Methyltransferase 1 and Plays an Anti-Leukemic Role in Acute Myeloid Leukemia
    Article Snippet: The primers for amplification of p15INK4B and its bisulfite-converted promoter region, and also for DNMT1 and its Sp1-binding promoter region, were purchased from Sigma-Aldrich or Integrated DNA Technology (IDT, Coralville, IA). .. M. SssI methylase, s-adenosylmethionine (SAM) (3.2 mM) and 10X incubation buffer were purchased from New England Biolabs (Ipswich, MA). .. DNMT1 antibody was purchased from New England Biolabs, β–actin antibody from Aldrich-Sigma, and GAPDH antibody (HRP Conjugate) from Cell Signaling Technology (Danvers, MA).

    Article Title: MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma
    Article Snippet: Prior to bisulfite conversion, fully methylated positive controls were also generated by incubating samples with S-Adenosyl methionine (SAM) and DNA methyltransferase (New England Biolabs) at 37°C for 2 hr. .. Prior to bisulfite conversion, fully methylated positive controls were also generated by incubating samples with S-Adenosyl methionine (SAM) and DNA methyltransferase (New England Biolabs) at 37°C for 2 hr.

    Article Title: Turn-on DNA Damage Sensors for the Direct Detection of 8-Oxoguanine and Photoproducts in Native DNA
    Article Snippet: All designed DNA targets were annealed in 1× annealing buffer (10 mM Tris-HCl, pH 7.9, 150 mM NaCl, 10 mM MgCl2 , 1mM DTT) using the following procedure: heating to 95 °C for 7 min, cooling to 56 °C at a rate of 0.1 °C sec−1 , equilibrating at 56 °C for 5 min, cooling to 25 °C at a rate of 0.1 °C sec−1 and finally equilibrating at 25 °C for 10 min using a Labnet Multi Gene II thermocycler. .. A pETDuet-derived plasmid (7667 bp) was fully methylated, generating pETm, by incubation with S-adenosylmethionine and M.SssI CpG methyltransferase (New England Biolabs) according to the manufacturer’s protocol. .. The extent of protection was determined by exposure to the methylation sensitive endonuclease BstUI.

    Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation
    Article Snippet: Cloned vector was then methylated in vitro utilizing Ss1 methylases (New England Biolabs, Frankfurt, Germany) according to the manufacturer's instruction. .. Briefly, 1 μg of plasmid DNA was added to a reaction containing CpG methyltransferase (Sss1, 4 units/μl) in the presence of 160 μ m S -adenosylmethionine (New England Biolabs) and incubated for 4 h at 37 °C, and S -adenosylmethionine was replenished after every 2 h. Unmethylated control reaction contained the NPC1L1 promoter construct and methylases but not S -adenosylmethionine. .. Plasmid DNA was then purified by using Qiagen miniprep kit and quantified using a spectrophotometer.

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori
    Article Snippet: The results were evaluated using a PhosphorImager (Molecular Dynamics Inc.). .. To investigate methylation and protection by the recombinant protein, we incubated 1 μg of a PCR fragment containing one GANTC site (778 bp, amplified with 1351GANTCF and 1351GANTCR) (Table ) with NEB2 buffer, S -adenosylmethionine (New England BioLabs), and different M.HpyAIV concentrations (0, 200, 400, 800, and 1,200 nM) and performed incubation for 1 h at room temperature followed by protein inactivation at 95°C for 10 min. .. Samples were digested with HinfI overnight at 37°C.

    Article Title: Structure of the Arginine Methyltransferase PRMT5-MEP50 Reveals a Mechanism for Substrate Specificity
    Article Snippet: Peptides synthesized using spot synthesis were chemoselectively immobilized onto functionalized glass slides as described earlier . .. For activity assays 10 µg/mL PRMT5-MEP50 complex was incubated in KCl/HEPES buffer (100 mM KCl (pH 7.5), 20 mM HEPES, 1 mM EDTA, 0.1 mM DTT, and 10% glycerol) in presence or absence of 1.5 mM S-adenosylmethionine (New England Biolabs, Ipswich, MA). .. Reactions were carried out on peptide microarrays at 30°C for 12 hours in a humidity chamber.

    Article Title: CREB/ATF-dependent T cell receptor-induced FoxP3 gene expression: a role for DNA methylation
    Article Snippet: The PCR product was cloned using the TOPO TA Cloning kit (Invitrogen). .. For plasmid methylation, the +4,301 to +4,500 CpG island region was amplified by PCR, and 5 μg of PCR product was incubated with 80 U of SssI methylase and 640 μM S-adenosylmethionine (New England Biolabs) for 2 h at 37°C. .. DNA methylation was verified using AatII, which is a methylation-sensitive restriction enzyme.

    Article Title: Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping
    Article Snippet: Protein samples were diluted to their final concentration in 1× buffer solution (10 mM MgCl2 , 10 mM KCl, 10 mM Tris–HCl pH 7.9) in the presence of 100 μM S -adenosyl-l -methionine (AdoMet, New England Biolabs, MA, USA). .. DNA samples were diluted to a final concentration of ∼0.75 nM in 1× buffer solution.

    Article Title: Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule
    Article Snippet: Construction of the plasmids expressing WT M.SssI or C141S, purification of the enzymes ( ) and coupling of the 5′-TTTTTTTTTTTTTTTCTCTCTTTT-3′ TFO to M.SssI(C141S) were done as described( ). .. Plasmids were incubated with 5-fold molar excess of TFO−C141S, M.SssI, C141S, or TFO in a buffer containing 20 mM Tris, 50 mM NaCl, 10 mM MgCl2 , pH 7.9, with or without 640 μM S -adenosylmethionine (SAM) (New England Biolabs, Ipswich, MA) at 30 °C. .. The reaction was terminated after 20 h by heat inactivation at 65 °C for 20 min, and plasmids were purified by Qiagen PCR purification kit (Qiagen, Benelux B.V., Venlo, The Netherlands).

    Luciferase:

    Article Title: Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells
    Article Snippet: Paragraph title: Cloning of the upstream region of precursor mir-142 , construction of expression constructs, in vitro DNA methylation and luciferase experiments ... 18 µg of pCpGL/2031 were in vitro methylated using the CpG methyltransferase M.SssI (4 units/µg), in the presence of 640 µM S-Adenosylmethionine (both from New England Biolabs) for 3 hours at 37°C and designated pCpGL/2031_M.SssI.

    Article Title: Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation
    Article Snippet: The methylated plasmids (Met-pGL3-446/-396) were generated by incubating 1 μg of plasmid DNA (pGL3 -446/-396) with 10 units Hpall methyltransferase in 10 μl 1 × Hpall methyltransferase buffer and 80 μM S-adenosylmethionine according to the manufacturer’s protocols (New England Biolabs, Inc.). .. The methylated plasmid DNA was transfected into 293T cell lines in parallel with the unmethylated pGL-446/-396, respectively.

    Activity Assay:

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy
    Article Snippet: Paragraph title: PRC2 histone methyltransferase activity assay ... Reactions were carried out in a volume of 50 μl and contained 2 pmol nucleosome (New England Biolabs, MA, USA), 2 pmol PRC2 complex, 10 mM HEPES (pH 7.9), 0.25 mM EDTA, 200 mM NaCl, 10% glycerol, 2 mM dithiothreitol, 2.5 mM MgCl2 and 0.8 μM S-adenosyl-methionine (New England Biolabs, MA, USA) with and without 10 nM Chaer RNA or 200 nM Ezh2 inhibitor GSK126 (Merck Millipore, Darmstadt, Germany).

    Article Title: Structure of the Arginine Methyltransferase PRMT5-MEP50 Reveals a Mechanism for Substrate Specificity
    Article Snippet: Peptides synthesized using spot synthesis were chemoselectively immobilized onto functionalized glass slides as described earlier . .. For activity assays 10 µg/mL PRMT5-MEP50 complex was incubated in KCl/HEPES buffer (100 mM KCl (pH 7.5), 20 mM HEPES, 1 mM EDTA, 0.1 mM DTT, and 10% glycerol) in presence or absence of 1.5 mM S-adenosylmethionine (New England Biolabs, Ipswich, MA). .. Reactions were carried out on peptide microarrays at 30°C for 12 hours in a humidity chamber.

    Article Title: Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule
    Article Snippet: Plasmids were incubated with 5-fold molar excess of TFO−C141S, M.SssI, C141S, or TFO in a buffer containing 20 mM Tris, 50 mM NaCl, 10 mM MgCl2 , pH 7.9, with or without 640 μM S -adenosylmethionine (SAM) (New England Biolabs, Ipswich, MA) at 30 °C. .. The reaction was terminated after 20 h by heat inactivation at 65 °C for 20 min, and plasmids were purified by Qiagen PCR purification kit (Qiagen, Benelux B.V., Venlo, The Netherlands).

    Article Title: Modulation of Dnmt3b function in vitro by interactions with Dnmt3L, Dnmt3a and Dnmt3b splice variants
    Article Snippet: Films with pull down and input were scanned and the composite images shown generated without further computerized image adjustment (e.g. alterations in contrast, brightness, etc.). .. Activity assay conditions, adapted from Takeshima et al . , were 20 mM Tris (pH 7.4), 5 mM EDTA, 25 mM NaCl, 10% glycerol, 200 µM DTT, 80 µM S -adenosyl-l -methionine (SAM, New England Biolabs) and 1.5 µg pSL301 plasmid containing the SAT2 sequence. .. The reaction mixture was incubated at 37°C for 7 h, with fresh SAM added after the first 4 h. For reactions comparing enzyme activity with and without Dnmt3L, 0.15 nmol of each purified methyltransferase was mixed in equimolar concentrations.

    Article Title: Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation
    Article Snippet: The methylated plasmids (Met-pGL3-446/-396) were generated by incubating 1 μg of plasmid DNA (pGL3 -446/-396) with 10 units Hpall methyltransferase in 10 μl 1 × Hpall methyltransferase buffer and 80 μM S-adenosylmethionine according to the manufacturer’s protocols (New England Biolabs, Inc.). .. The methylated plasmid DNA was transfected into 293T cell lines in parallel with the unmethylated pGL-446/-396, respectively.

    Expressing:

    Article Title: Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells
    Article Snippet: Paragraph title: Cloning of the upstream region of precursor mir-142 , construction of expression constructs, in vitro DNA methylation and luciferase experiments ... 18 µg of pCpGL/2031 were in vitro methylated using the CpG methyltransferase M.SssI (4 units/µg), in the presence of 640 µM S-Adenosylmethionine (both from New England Biolabs) for 3 hours at 37°C and designated pCpGL/2031_M.SssI.

    Article Title: Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule
    Article Snippet: Construction of the plasmids expressing WT M.SssI or C141S, purification of the enzymes ( ) and coupling of the 5′-TTTTTTTTTTTTTTTCTCTCTTTT-3′ TFO to M.SssI(C141S) were done as described( ). .. Plasmids were incubated with 5-fold molar excess of TFO−C141S, M.SssI, C141S, or TFO in a buffer containing 20 mM Tris, 50 mM NaCl, 10 mM MgCl2 , pH 7.9, with or without 640 μM S -adenosylmethionine (SAM) (New England Biolabs, Ipswich, MA) at 30 °C.

    Modification:

    Article Title: The Drosophila G9a gene encodes a multi-catalytic histone methyltransferase required for normal development
    Article Snippet: Methylation reactions were carried out as described above with 0.5 µg of histone H3 or H4 and 250 µM of S-Adenosylmethionine (New England BioLabs). .. The reaction was stopped by addition of SDS–PAGE loading buffer and the histones were separated by 15% SDS–PAGE.

    Article Title: Integrative Analysis of Hereditary Nonpolyposis Colorectal Cancer: the Contribution of Allele-Specific Expression and Other Assays to Diagnostic Algorithms
    Article Snippet: Bisulphite DNA conversion was performed in in 44 cases negative at initial screening for pathogenic sequence variants and genomic rearrangements using the Imprinting DNA modification kit (SIGMA, Saint Louis, MO), according to the manufacturer's instructions. .. Positive controls for MSH2 promoter methylation were obtained using control DNAs that had been universally methylated using S-adenosylmethionine (SAM) and M.SssI CpG methyltrasferase (New England BioLabs, Ipswich, MA).

    Transformation Assay:

    Article Title: Twin-Arginine Translocation System in Helicobacter pylori: TatC, but Not TatB, Is Essential for Viability
    Article Snippet: Plasmid pSLB495 was methylated in the presence of S -adenosylmethionine (New England Biolabs, Ipswich, MA) and cell-free extracts from H. pylori parental strain 43504, as described by Boneca et al. ( ). .. Methylated plasmid pSLB495 was then introduced into strain 43504, and chloramphenicol-resistant mutants were isolated.

    High Performance Liquid Chromatography:

    Article Title: Curcumin Down-Regulates DNA Methyltransferase 1 and Plays an Anti-Leukemic Role in Acute Myeloid Leukemia
    Article Snippet: Curcumin, methanol, acetonitrile (HPLC grade), ammonium formate, ammonium acetate, ammonium bicarbonate, 5-methyl-2-deoxycytidine (5mdC), 2-deoxycytidine (2dC), 2-deoxyguanosine (2dG), nucleophosphatase (NP1), snake venom phosphatase (SVP), alkaline phosphatase (AP), deoxynucleotide triphosphate (2.5 mM), AmpliTaqGold polymerase, and 10X PCR buffer were purchased from Sigma-Aldrich (St. Louis, MO). .. M. SssI methylase, s-adenosylmethionine (SAM) (3.2 mM) and 10X incubation buffer were purchased from New England Biolabs (Ipswich, MA).

    Transfection:

    Article Title: Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells
    Article Snippet: 18 µg of pCpGL/2031 were in vitro methylated using the CpG methyltransferase M.SssI (4 units/µg), in the presence of 640 µM S-Adenosylmethionine (both from New England Biolabs) for 3 hours at 37°C and designated pCpGL/2031_M.SssI. .. 18 µg of pCpGL/2031 were in vitro methylated using the CpG methyltransferase M.SssI (4 units/µg), in the presence of 640 µM S-Adenosylmethionine (both from New England Biolabs) for 3 hours at 37°C and designated pCpGL/2031_M.SssI.

    Article Title: CREB/ATF-dependent T cell receptor-induced FoxP3 gene expression: a role for DNA methylation
    Article Snippet: For plasmid methylation, the +4,301 to +4,500 CpG island region was amplified by PCR, and 5 μg of PCR product was incubated with 80 U of SssI methylase and 640 μM S-adenosylmethionine (New England Biolabs) for 2 h at 37°C. .. DNA methylation was verified using AatII, which is a methylation-sensitive restriction enzyme.

    Article Title: Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation
    Article Snippet: Paragraph title: In vitro DNA methylation and transient transfection ... The methylated plasmids (Met-pGL3-446/-396) were generated by incubating 1 μg of plasmid DNA (pGL3 -446/-396) with 10 units Hpall methyltransferase in 10 μl 1 × Hpall methyltransferase buffer and 80 μM S-adenosylmethionine according to the manufacturer’s protocols (New England Biolabs, Inc.).

    Southern Blot:

    Article Title: SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC
    Article Snippet: Methylation was assayed by digesting an aliquot of the plasmid with Hph I, followed by agarose gel electrophoresis and Southern blotting. .. PCR products (2μg) purified using a QIAquick PCR purification kit were treated with 8 units of dam methylase and 800μM S-adenosylmethionine (New England Biolabs) at 37°C overnight.

    Transferring:

    Article Title: The Drosophila G9a gene encodes a multi-catalytic histone methyltransferase required for normal development
    Article Snippet: Methylation reactions were carried out as described above with 0.5 µg of histone H3 or H4 and 250 µM of S-Adenosylmethionine (New England BioLabs). .. H3 and H4 were digested over night with 100 ng of sequencing-grade trypsin (Promega) in a total volume of 40 µl according to manufacturer's protocol.

    Introduce:

    Article Title: Turn-on DNA Damage Sensors for the Direct Detection of 8-Oxoguanine and Photoproducts in Native DNA
    Article Snippet: A pETDuet-derived plasmid (7667 bp) was fully methylated, generating pETm, by incubation with S-adenosylmethionine and M.SssI CpG methyltransferase (New England Biolabs) according to the manufacturer’s protocol. .. The extent of protection was determined by exposure to the methylation sensitive endonuclease BstUI.

    Generated:

    Article Title: MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma
    Article Snippet: The samples were incubated at various denaturation and annealing cycles for approximately 5 hr, which were then processed following manufacturer’s instructions. .. Prior to bisulfite conversion, fully methylated positive controls were also generated by incubating samples with S-Adenosyl methionine (SAM) and DNA methyltransferase (New England Biolabs) at 37°C for 2 hr. .. Direct bisulfite sequencing was carried out to determine the methylation status of MST4 at single base resolution using gel-purified CoBRA PCR products.

    Article Title: SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC
    Article Snippet: Hemimethylated linear templates were generated from fully methylated PCR products by performing one additional PCR cycle. oriC was first amplified with 22 bp PCR primers annealing at position −184 and 343 (numbered as in ), to generated a 527 bp fragment. .. PCR products (2μg) purified using a QIAquick PCR purification kit were treated with 8 units of dam methylase and 800μM S-adenosylmethionine (New England Biolabs) at 37°C overnight.

    Article Title: Structure of the Arginine Methyltransferase PRMT5-MEP50 Reveals a Mechanism for Substrate Specificity
    Article Snippet: A library of 20-mer peptides spanning the sequences of histones H2A (P0C0S8), H2B (P62807), H3 (P68431) and H4 (P62805) was generated including single known modifications and in various combinations (sequences available at www.jpt.com ). .. For activity assays 10 µg/mL PRMT5-MEP50 complex was incubated in KCl/HEPES buffer (100 mM KCl (pH 7.5), 20 mM HEPES, 1 mM EDTA, 0.1 mM DTT, and 10% glycerol) in presence or absence of 1.5 mM S-adenosylmethionine (New England Biolabs, Ipswich, MA).

    Article Title: Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation
    Article Snippet: Finally, the biotin-DNA was detected by chemiluminescence and visualized by Biospectrum® 510 Imaging System (Upland, CA, USA). .. The methylated plasmids (Met-pGL3-446/-396) were generated by incubating 1 μg of plasmid DNA (pGL3 -446/-396) with 10 units Hpall methyltransferase in 10 μl 1 × Hpall methyltransferase buffer and 80 μM S-adenosylmethionine according to the manufacturer’s protocols (New England Biolabs, Inc.). .. Reactions were carried out at 37 °C 1 h followed by 15 min at 65 °C to inactivate the methylase, purified by polyacrylamide gel electrophoresis.

    Sequencing:

    Article Title: The Drosophila G9a gene encodes a multi-catalytic histone methyltransferase required for normal development
    Article Snippet: Methylation reactions were carried out as described above with 0.5 µg of histone H3 or H4 and 250 µM of S-Adenosylmethionine (New England BioLabs). .. The Coomassie stained bands corresponding to H3 and H4 were excised and subjected to chemical modification to derive free amino groups of lysine residues as described previously ( ).

    Article Title: MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma
    Article Snippet: Prior to bisulfite conversion, fully methylated positive controls were also generated by incubating samples with S-Adenosyl methionine (SAM) and DNA methyltransferase (New England Biolabs) at 37°C for 2 hr. .. Direct bisulfite sequencing was carried out to determine the methylation status of MST4 at single base resolution using gel-purified CoBRA PCR products.

    Article Title: Rational protein sequence diversification by multi-codon scanning mutagenesis
    Article Snippet: Paragraph title: 3.3. Introduction of new codons and “anchor” sequence ... Incubate the reaction at 30 °C for 4.5 h. Heat-inactivate the reaction at 65 °C for 20 min. Digest the purified PCR product of two and three codon libraries with Bsg I in a 50 μL reaction containing 500 ng DNA, 6 U Bsg I (NEB), 80 μM S -adenosylmethionine (SAM) and 1× buffer 4 (NEB) ( see ).

    Article Title: Integrative Analysis of Hereditary Nonpolyposis Colorectal Cancer: the Contribution of Allele-Specific Expression and Other Assays to Diagnostic Algorithms
    Article Snippet: Bisulphite DNA conversion was performed in in 44 cases negative at initial screening for pathogenic sequence variants and genomic rearrangements using the Imprinting DNA modification kit (SIGMA, Saint Louis, MO), according to the manufacturer's instructions. .. Positive controls for MSH2 promoter methylation were obtained using control DNAs that had been universally methylated using S-adenosylmethionine (SAM) and M.SssI CpG methyltrasferase (New England BioLabs, Ipswich, MA).

    Article Title: Modulation of Dnmt3b function in vitro by interactions with Dnmt3L, Dnmt3a and Dnmt3b splice variants
    Article Snippet: Films with pull down and input were scanned and the composite images shown generated without further computerized image adjustment (e.g. alterations in contrast, brightness, etc.). .. Activity assay conditions, adapted from Takeshima et al . , were 20 mM Tris (pH 7.4), 5 mM EDTA, 25 mM NaCl, 10% glycerol, 200 µM DTT, 80 µM S -adenosyl-l -methionine (SAM, New England Biolabs) and 1.5 µg pSL301 plasmid containing the SAT2 sequence. .. The reaction mixture was incubated at 37°C for 7 h, with fresh SAM added after the first 4 h. For reactions comparing enzyme activity with and without Dnmt3L, 0.15 nmol of each purified methyltransferase was mixed in equimolar concentrations.

    Recombinant:

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori
    Article Snippet: The results were evaluated using a PhosphorImager (Molecular Dynamics Inc.). .. To investigate methylation and protection by the recombinant protein, we incubated 1 μg of a PCR fragment containing one GANTC site (778 bp, amplified with 1351GANTCF and 1351GANTCR) (Table ) with NEB2 buffer, S -adenosylmethionine (New England BioLabs), and different M.HpyAIV concentrations (0, 200, 400, 800, and 1,200 nM) and performed incubation for 1 h at room temperature followed by protein inactivation at 95°C for 10 min. .. Samples were digested with HinfI overnight at 37°C.

    Combined Bisulfite Restriction Analysis Assay:

    Article Title: MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma
    Article Snippet: Bisulfite conversion of genomic DNA was carried out prior to CoBRA analyses using EpiTect Bisulfite conversion kit (Qiagen). .. Prior to bisulfite conversion, fully methylated positive controls were also generated by incubating samples with S-Adenosyl methionine (SAM) and DNA methyltransferase (New England Biolabs) at 37°C for 2 hr.

    Fluorescence:

    Article Title: Structure of the Arginine Methyltransferase PRMT5-MEP50 Reveals a Mechanism for Substrate Specificity
    Article Snippet: For activity assays 10 µg/mL PRMT5-MEP50 complex was incubated in KCl/HEPES buffer (100 mM KCl (pH 7.5), 20 mM HEPES, 1 mM EDTA, 0.1 mM DTT, and 10% glycerol) in presence or absence of 1.5 mM S-adenosylmethionine (New England Biolabs, Ipswich, MA). .. The microarrays were incubated with anti-H4R3me2s rabbit polyclonal antibody (Millipore, #07–947) followed by washing and incubation with fluorescently labeled secondary antibody (DL649-anti-rabbit IgG; Pierce, #35565).

    Methylation:

    Article Title: The Drosophila G9a gene encodes a multi-catalytic histone methyltransferase required for normal development
    Article Snippet: Exposure time for autoradiograph was 1 and 2 days. .. Methylation reactions were carried out as described above with 0.5 µg of histone H3 or H4 and 250 µM of S-Adenosylmethionine (New England BioLabs). .. The reaction was stopped by addition of SDS–PAGE loading buffer and the histones were separated by 15% SDS–PAGE.

    Article Title: Dietary Flavones as Dual Inhibitors of DNA Methyltransferases and Histone Methyltransferases
    Article Snippet: The substrate DNA for an in vitro methylation assay was a 721 bp fragment (-428/+243 relative to the initiation codon), which was amplified from RWPE-1 cells within the promoter region of the human GSTP1 gene. .. The methylation reactions were carried out in 1X M.SssI buffer with 160 mM SAM S-adenosylmethionine (supplied with M.SssI by New England Biolabs). .. For methylation, 1 μg of the purified GSTP1 promoter PCR product was incubated with 10 U of M.SssI methyltransferase enzyme (New England Biolabs, Ipswich, MA, USA) with or without flavones in 1X MSss1 buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM EDTA and 1 mM dithiothreitol), pH 8.0, for 3 h at 37°C in 50 μl of reaction volume.

    Article Title: MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma
    Article Snippet: The samples were incubated at various denaturation and annealing cycles for approximately 5 hr, which were then processed following manufacturer’s instructions. .. Prior to bisulfite conversion, fully methylated positive controls were also generated by incubating samples with S-Adenosyl methionine (SAM) and DNA methyltransferase (New England Biolabs) at 37°C for 2 hr. .. Direct bisulfite sequencing was carried out to determine the methylation status of MST4 at single base resolution using gel-purified CoBRA PCR products.

    Article Title: Synthesis of 5? cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N7)-methyl transferase
    Article Snippet: Betaplate Scint (Wallac) scintillation fluid was added, and the methylation of RNA substrates was measured in counts per minute (c.p.m.) using a Wallac 1450 MicroBeta TriLux Liquid Scintillation Counter. .. Methylation of purified Gppp-RNAs ( 2–4 ), ( 9–10 ) (∼70 nanomol) to give 7m GpppN-RNAs ( 12–15 ) and 7m GpppN2′-Om -RNA ( 16 ) was carried out using 0.25 μM N 7 -hMTase and 0.4 mM S -adenosylmethionine (New England Biolabs) in 40 mM Tris-HCl, pH 8, with 5 mM dithiothreitol in a 1725-μL reaction volume (Gppp-RNAs at a final concentration of 40 μM) at 30°C. .. N 7 -methylation was monitored using IEX-HPLC analysis of an aliquot from the reaction mixture and passed through ZipTip C18 (see procedure below) prior analysis to remove protein.

    Article Title: Turn-on DNA Damage Sensors for the Direct Detection of 8-Oxoguanine and Photoproducts in Native DNA
    Article Snippet: All designed DNA targets were annealed in 1× annealing buffer (10 mM Tris-HCl, pH 7.9, 150 mM NaCl, 10 mM MgCl2 , 1mM DTT) using the following procedure: heating to 95 °C for 7 min, cooling to 56 °C at a rate of 0.1 °C sec−1 , equilibrating at 56 °C for 5 min, cooling to 25 °C at a rate of 0.1 °C sec−1 and finally equilibrating at 25 °C for 10 min using a Labnet Multi Gene II thermocycler. .. A pETDuet-derived plasmid (7667 bp) was fully methylated, generating pETm, by incubation with S-adenosylmethionine and M.SssI CpG methyltransferase (New England Biolabs) according to the manufacturer’s protocol. .. The extent of protection was determined by exposure to the methylation sensitive endonuclease BstUI.

    Article Title: SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC
    Article Snippet: Hemimethylated linear templates were generated from fully methylated PCR products by performing one additional PCR cycle. oriC was first amplified with 22 bp PCR primers annealing at position −184 and 343 (numbered as in ), to generated a 527 bp fragment. .. PCR products (2μg) purified using a QIAquick PCR purification kit were treated with 8 units of dam methylase and 800μM S-adenosylmethionine (New England Biolabs) at 37°C overnight.

    Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation
    Article Snippet: Paragraph title: Cloning and in Vitro Methylation of pCpG Free-L1 Vector ... Briefly, 1 μg of plasmid DNA was added to a reaction containing CpG methyltransferase (Sss1, 4 units/μl) in the presence of 160 μ m S -adenosylmethionine (New England Biolabs) and incubated for 4 h at 37 °C, and S -adenosylmethionine was replenished after every 2 h. Unmethylated control reaction contained the NPC1L1 promoter construct and methylases but not S -adenosylmethionine.

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori
    Article Snippet: The results were evaluated using a PhosphorImager (Molecular Dynamics Inc.). .. To investigate methylation and protection by the recombinant protein, we incubated 1 μg of a PCR fragment containing one GANTC site (778 bp, amplified with 1351GANTCF and 1351GANTCR) (Table ) with NEB2 buffer, S -adenosylmethionine (New England BioLabs), and different M.HpyAIV concentrations (0, 200, 400, 800, and 1,200 nM) and performed incubation for 1 h at room temperature followed by protein inactivation at 95°C for 10 min. .. Samples were digested with HinfI overnight at 37°C.

    Article Title: Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells
    Article Snippet: The pCpGL-basic with the cloned upstream region was designated pCpGL/2031. .. 18 µg of pCpGL/2031 were in vitro methylated using the CpG methyltransferase M.SssI (4 units/µg), in the presence of 640 µM S-Adenosylmethionine (both from New England Biolabs) for 3 hours at 37°C and designated pCpGL/2031_M.SssI. .. The unmethylated control reporter vector (pCpGL/2031_mock) was treated as above but without methyltransferase.

    Article Title: Integrative Analysis of Hereditary Nonpolyposis Colorectal Cancer: the Contribution of Allele-Specific Expression and Other Assays to Diagnostic Algorithms
    Article Snippet: Screening for germline MSH2 promoter methylation was conducted by MSP using primers specific for methylated and unmethylated alleles, as described by Chan et al [ ]. .. Positive controls for MSH2 promoter methylation were obtained using control DNAs that had been universally methylated using S-adenosylmethionine (SAM) and M.SssI CpG methyltrasferase (New England BioLabs, Ipswich, MA). .. ASE analyses of MSH2 , MLH1 or MSH6 were performed by primer extension in patients with available RNA and heterozygous for at least one allelic marker, independently from their mutational status.

    Article Title: Twin-Arginine Translocation System in Helicobacter pylori: TatC, but Not TatB, Is Essential for Viability
    Article Snippet: In this plasmid, the tatC gene is under the control of a (IPTG-inducible) Ptac promoter. .. Plasmid pSLB495 was methylated in the presence of S -adenosylmethionine (New England Biolabs, Ipswich, MA) and cell-free extracts from H. pylori parental strain 43504, as described by Boneca et al. ( ). .. Methylated plasmid pSLB495 was then introduced into strain 43504, and chloramphenicol-resistant mutants were isolated.

    Article Title: CREB/ATF-dependent T cell receptor-induced FoxP3 gene expression: a role for DNA methylation
    Article Snippet: The PCR product was cloned using the TOPO TA Cloning kit (Invitrogen). .. For plasmid methylation, the +4,301 to +4,500 CpG island region was amplified by PCR, and 5 μg of PCR product was incubated with 80 U of SssI methylase and 640 μM S-adenosylmethionine (New England Biolabs) for 2 h at 37°C. .. DNA methylation was verified using AatII, which is a methylation-sensitive restriction enzyme.

    Article Title: Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule
    Article Snippet: Paragraph title: Methylation of Plasmids with TFO−C141S ... Plasmids were incubated with 5-fold molar excess of TFO−C141S, M.SssI, C141S, or TFO in a buffer containing 20 mM Tris, 50 mM NaCl, 10 mM MgCl2 , pH 7.9, with or without 640 μM S -adenosylmethionine (SAM) (New England Biolabs, Ipswich, MA) at 30 °C.

    Article Title: Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation
    Article Snippet: Finally, the biotin-DNA was detected by chemiluminescence and visualized by Biospectrum® 510 Imaging System (Upland, CA, USA). .. The methylated plasmids (Met-pGL3-446/-396) were generated by incubating 1 μg of plasmid DNA (pGL3 -446/-396) with 10 units Hpall methyltransferase in 10 μl 1 × Hpall methyltransferase buffer and 80 μM S-adenosylmethionine according to the manufacturer’s protocols (New England Biolabs, Inc.). .. Reactions were carried out at 37 °C 1 h followed by 15 min at 65 °C to inactivate the methylase, purified by polyacrylamide gel electrophoresis.

    Mutagenesis:

    Article Title: Twin-Arginine Translocation System in Helicobacter pylori: TatC, but Not TatB, Is Essential for Viability
    Article Snippet: Paragraph title: Construction of a conditional tatC mutant. ... Plasmid pSLB495 was methylated in the presence of S -adenosylmethionine (New England Biolabs, Ipswich, MA) and cell-free extracts from H. pylori parental strain 43504, as described by Boneca et al. ( ).

    Isolation:

    Article Title: MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma
    Article Snippet: Briefly, 300 ng of genomic DNA isolated from GSCs was mixed up with 85 μl bisulfite mix and 15 μl DNA protect buffer making up a total volume of 140 μl with RNase free water. .. Prior to bisulfite conversion, fully methylated positive controls were also generated by incubating samples with S-Adenosyl methionine (SAM) and DNA methyltransferase (New England Biolabs) at 37°C for 2 hr.

    Article Title: SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC
    Article Snippet: Hemimethylated supercoiled templates were isolated from E. coli PC2 dnaC2 (ts) harboring minichromosome pAL49 after a temperature shift to align replication from oriC . .. PCR products (2μg) purified using a QIAquick PCR purification kit were treated with 8 units of dam methylase and 800μM S-adenosylmethionine (New England Biolabs) at 37°C overnight.

    Article Title: Twin-Arginine Translocation System in Helicobacter pylori: TatC, but Not TatB, Is Essential for Viability
    Article Snippet: Plasmid pSLB495 was methylated in the presence of S -adenosylmethionine (New England Biolabs, Ipswich, MA) and cell-free extracts from H. pylori parental strain 43504, as described by Boneca et al. ( ). .. The resulting strain, SLB1308, was transformed with plasmid pSLB137 (suicide vector harboring ΔtatC ::aphA3 [see above and ]) in the presence of Cm (8 µg/ml) and IPTG (1 mM).

    Labeling:

    Article Title: Structure of the Arginine Methyltransferase PRMT5-MEP50 Reveals a Mechanism for Substrate Specificity
    Article Snippet: For activity assays 10 µg/mL PRMT5-MEP50 complex was incubated in KCl/HEPES buffer (100 mM KCl (pH 7.5), 20 mM HEPES, 1 mM EDTA, 0.1 mM DTT, and 10% glycerol) in presence or absence of 1.5 mM S-adenosylmethionine (New England Biolabs, Ipswich, MA). .. Detection of methyltransferase activity was performed in a Tecan HS4800 microarray processing station .

    Purification:

    Article Title: Curcumin Down-Regulates DNA Methyltransferase 1 and Plays an Anti-Leukemic Role in Acute Myeloid Leukemia
    Article Snippet: Decitabine was obtained from the National Cancer Institute and used without further purification. .. M. SssI methylase, s-adenosylmethionine (SAM) (3.2 mM) and 10X incubation buffer were purchased from New England Biolabs (Ipswich, MA).

    Article Title: Dietary Flavones as Dual Inhibitors of DNA Methyltransferases and Histone Methyltransferases
    Article Snippet: The methylation reactions were carried out in 1X M.SssI buffer with 160 mM SAM S-adenosylmethionine (supplied with M.SssI by New England Biolabs). .. For methylation, 1 μg of the purified GSTP1 promoter PCR product was incubated with 10 U of M.SssI methyltransferase enzyme (New England Biolabs, Ipswich, MA, USA) with or without flavones in 1X MSss1 buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM EDTA and 1 mM dithiothreitol), pH 8.0, for 3 h at 37°C in 50 μl of reaction volume.

    Article Title: MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma
    Article Snippet: Prior to bisulfite conversion, fully methylated positive controls were also generated by incubating samples with S-Adenosyl methionine (SAM) and DNA methyltransferase (New England Biolabs) at 37°C for 2 hr. .. Prior to bisulfite conversion, fully methylated positive controls were also generated by incubating samples with S-Adenosyl methionine (SAM) and DNA methyltransferase (New England Biolabs) at 37°C for 2 hr.

    Article Title: Synthesis of 5? cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N7)-methyl transferase
    Article Snippet: Betaplate Scint (Wallac) scintillation fluid was added, and the methylation of RNA substrates was measured in counts per minute (c.p.m.) using a Wallac 1450 MicroBeta TriLux Liquid Scintillation Counter. .. Methylation of purified Gppp-RNAs ( 2–4 ), ( 9–10 ) (∼70 nanomol) to give 7m GpppN-RNAs ( 12–15 ) and 7m GpppN2′-Om -RNA ( 16 ) was carried out using 0.25 μM N 7 -hMTase and 0.4 mM S -adenosylmethionine (New England Biolabs) in 40 mM Tris-HCl, pH 8, with 5 mM dithiothreitol in a 1725-μL reaction volume (Gppp-RNAs at a final concentration of 40 μM) at 30°C. .. N 7 -methylation was monitored using IEX-HPLC analysis of an aliquot from the reaction mixture and passed through ZipTip C18 (see procedure below) prior analysis to remove protein.

    Article Title: SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC
    Article Snippet: Hemimethylated linear templates were generated from fully methylated PCR products by performing one additional PCR cycle. oriC was first amplified with 22 bp PCR primers annealing at position −184 and 343 (numbered as in ), to generated a 527 bp fragment. .. PCR products (2μg) purified using a QIAquick PCR purification kit were treated with 8 units of dam methylase and 800μM S-adenosylmethionine (New England Biolabs) at 37°C overnight. .. The methylated DNA was amplified for one additional cycle and hemimethylation was analyzed by Hph1 digestion as described above except a 199 bp derived from an Hph I digestion of oriC DNA was used as probe.

    Article Title: Rational protein sequence diversification by multi-codon scanning mutagenesis
    Article Snippet: Digest the purified PCR product of one codon library with Bpm I in a 50 μL reaction containing 500 ng DNA, 1 U FastDigest Bpm I (Fermentas) and 1×FastDigest buffer (Fermentas) ( see ). .. Incubate the reaction at 30 °C for 4.5 h. Heat-inactivate the reaction at 65 °C for 20 min. Digest the purified PCR product of two and three codon libraries with Bsg I in a 50 μL reaction containing 500 ng DNA, 6 U Bsg I (NEB), 80 μM S -adenosylmethionine (SAM) and 1× buffer 4 (NEB) ( see ). .. Incubate the reaction at 37 °C for 4.5 h. Heat-inactivate the reaction at 65 °C for 20 min. 5.

    Article Title: Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells
    Article Snippet: 18 µg of pCpGL/2031 were in vitro methylated using the CpG methyltransferase M.SssI (4 units/µg), in the presence of 640 µM S-Adenosylmethionine (both from New England Biolabs) for 3 hours at 37°C and designated pCpGL/2031_M.SssI. .. The unmethylated control reporter vector (pCpGL/2031_mock) was treated as above but without methyltransferase.

    Article Title: CREB/ATF-dependent T cell receptor-induced FoxP3 gene expression: a role for DNA methylation
    Article Snippet: For plasmid methylation, the +4,301 to +4,500 CpG island region was amplified by PCR, and 5 μg of PCR product was incubated with 80 U of SssI methylase and 640 μM S-adenosylmethionine (New England Biolabs) for 2 h at 37°C. .. DNA methylation was verified using AatII, which is a methylation-sensitive restriction enzyme.

    Article Title: Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping
    Article Snippet: Protein samples were diluted to their final concentration in 1× buffer solution (10 mM MgCl2 , 10 mM KCl, 10 mM Tris–HCl pH 7.9) in the presence of 100 μM S -adenosyl-l -methionine (AdoMet, New England Biolabs, MA, USA). .. Protein samples were diluted to their final concentration in 1× buffer solution (10 mM MgCl2 , 10 mM KCl, 10 mM Tris–HCl pH 7.9) in the presence of 100 μM S -adenosyl-l -methionine (AdoMet, New England Biolabs, MA, USA).

    Article Title: Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule
    Article Snippet: Construction of the plasmids expressing WT M.SssI or C141S, purification of the enzymes ( ) and coupling of the 5′-TTTTTTTTTTTTTTTCTCTCTTTT-3′ TFO to M.SssI(C141S) were done as described( ). .. Plasmids were incubated with 5-fold molar excess of TFO−C141S, M.SssI, C141S, or TFO in a buffer containing 20 mM Tris, 50 mM NaCl, 10 mM MgCl2 , pH 7.9, with or without 640 μM S -adenosylmethionine (SAM) (New England Biolabs, Ipswich, MA) at 30 °C.

    Article Title: Modulation of Dnmt3b function in vitro by interactions with Dnmt3L, Dnmt3a and Dnmt3b splice variants
    Article Snippet: Activity assay conditions, adapted from Takeshima et al . , were 20 mM Tris (pH 7.4), 5 mM EDTA, 25 mM NaCl, 10% glycerol, 200 µM DTT, 80 µM S -adenosyl-l -methionine (SAM, New England Biolabs) and 1.5 µg pSL301 plasmid containing the SAT2 sequence. .. The reaction mixture was incubated at 37°C for 7 h, with fresh SAM added after the first 4 h. For reactions comparing enzyme activity with and without Dnmt3L, 0.15 nmol of each purified methyltransferase was mixed in equimolar concentrations.

    Polymerase Chain Reaction:

    Article Title: Curcumin Down-Regulates DNA Methyltransferase 1 and Plays an Anti-Leukemic Role in Acute Myeloid Leukemia
    Article Snippet: Curcumin, methanol, acetonitrile (HPLC grade), ammonium formate, ammonium acetate, ammonium bicarbonate, 5-methyl-2-deoxycytidine (5mdC), 2-deoxycytidine (2dC), 2-deoxyguanosine (2dG), nucleophosphatase (NP1), snake venom phosphatase (SVP), alkaline phosphatase (AP), deoxynucleotide triphosphate (2.5 mM), AmpliTaqGold polymerase, and 10X PCR buffer were purchased from Sigma-Aldrich (St. Louis, MO). .. M. SssI methylase, s-adenosylmethionine (SAM) (3.2 mM) and 10X incubation buffer were purchased from New England Biolabs (Ipswich, MA).

    Article Title: Dietary Flavones as Dual Inhibitors of DNA Methyltransferases and Histone Methyltransferases
    Article Snippet: The methylation reactions were carried out in 1X M.SssI buffer with 160 mM SAM S-adenosylmethionine (supplied with M.SssI by New England Biolabs). .. For methylation, 1 μg of the purified GSTP1 promoter PCR product was incubated with 10 U of M.SssI methyltransferase enzyme (New England Biolabs, Ipswich, MA, USA) with or without flavones in 1X MSss1 buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM EDTA and 1 mM dithiothreitol), pH 8.0, for 3 h at 37°C in 50 μl of reaction volume.

    Article Title: MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma
    Article Snippet: Prior to bisulfite conversion, fully methylated positive controls were also generated by incubating samples with S-Adenosyl methionine (SAM) and DNA methyltransferase (New England Biolabs) at 37°C for 2 hr. .. Direct bisulfite sequencing was carried out to determine the methylation status of MST4 at single base resolution using gel-purified CoBRA PCR products.

    Article Title: SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC
    Article Snippet: Hemimethylated linear templates were generated from fully methylated PCR products by performing one additional PCR cycle. oriC was first amplified with 22 bp PCR primers annealing at position −184 and 343 (numbered as in ), to generated a 527 bp fragment. .. PCR products (2μg) purified using a QIAquick PCR purification kit were treated with 8 units of dam methylase and 800μM S-adenosylmethionine (New England Biolabs) at 37°C overnight. .. The methylated DNA was amplified for one additional cycle and hemimethylation was analyzed by Hph1 digestion as described above except a 199 bp derived from an Hph I digestion of oriC DNA was used as probe.

    Article Title: Rational protein sequence diversification by multi-codon scanning mutagenesis
    Article Snippet: Digest the purified PCR product of one codon library with Bpm I in a 50 μL reaction containing 500 ng DNA, 1 U FastDigest Bpm I (Fermentas) and 1×FastDigest buffer (Fermentas) ( see ). .. Incubate the reaction at 30 °C for 4.5 h. Heat-inactivate the reaction at 65 °C for 20 min. Digest the purified PCR product of two and three codon libraries with Bsg I in a 50 μL reaction containing 500 ng DNA, 6 U Bsg I (NEB), 80 μM S -adenosylmethionine (SAM) and 1× buffer 4 (NEB) ( see ). .. Incubate the reaction at 37 °C for 4.5 h. Heat-inactivate the reaction at 65 °C for 20 min. 5.

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori
    Article Snippet: The results were evaluated using a PhosphorImager (Molecular Dynamics Inc.). .. To investigate methylation and protection by the recombinant protein, we incubated 1 μg of a PCR fragment containing one GANTC site (778 bp, amplified with 1351GANTCF and 1351GANTCR) (Table ) with NEB2 buffer, S -adenosylmethionine (New England BioLabs), and different M.HpyAIV concentrations (0, 200, 400, 800, and 1,200 nM) and performed incubation for 1 h at room temperature followed by protein inactivation at 95°C for 10 min. .. Samples were digested with HinfI overnight at 37°C.

    Article Title: Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells
    Article Snippet: The 2,031 bp upstream region of pre-mir-142 was amplified using PCR and The AccuPrime Taq DNA Polymerase High Fidelity (Life Technologies) ( ) and T/A-cloned into the CpG-free pCpGL-basic luciferase reporter construct, which was a gift from Michael Rehli . .. 18 µg of pCpGL/2031 were in vitro methylated using the CpG methyltransferase M.SssI (4 units/µg), in the presence of 640 µM S-Adenosylmethionine (both from New England Biolabs) for 3 hours at 37°C and designated pCpGL/2031_M.SssI.

    Article Title: Integrative Analysis of Hereditary Nonpolyposis Colorectal Cancer: the Contribution of Allele-Specific Expression and Other Assays to Diagnostic Algorithms
    Article Snippet: Furthermore, we designed a control PCR in which the degenerate primer used for MSP was paired to a primer specific for unmethylated DNA ( ). .. Positive controls for MSH2 promoter methylation were obtained using control DNAs that had been universally methylated using S-adenosylmethionine (SAM) and M.SssI CpG methyltrasferase (New England BioLabs, Ipswich, MA).

    Article Title: Twin-Arginine Translocation System in Helicobacter pylori: TatC, but Not TatB, Is Essential for Viability
    Article Snippet: The NdeI- and BamHI-digested tatC PCR product obtained with primers TC7 and TC8 (see above and see and in the supplemental material) was ligated into similarly digested plasmid pILL2150 ( ) to generate plasmid pSLB495. .. Plasmid pSLB495 was methylated in the presence of S -adenosylmethionine (New England Biolabs, Ipswich, MA) and cell-free extracts from H. pylori parental strain 43504, as described by Boneca et al. ( ).

    Article Title: CREB/ATF-dependent T cell receptor-induced FoxP3 gene expression: a role for DNA methylation
    Article Snippet: The PCR product was cloned using the TOPO TA Cloning kit (Invitrogen). .. For plasmid methylation, the +4,301 to +4,500 CpG island region was amplified by PCR, and 5 μg of PCR product was incubated with 80 U of SssI methylase and 640 μM S-adenosylmethionine (New England Biolabs) for 2 h at 37°C. .. DNA methylation was verified using AatII, which is a methylation-sensitive restriction enzyme.

    Gel Extraction:

    Article Title: MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma
    Article Snippet: Prior to bisulfite conversion, fully methylated positive controls were also generated by incubating samples with S-Adenosyl methionine (SAM) and DNA methyltransferase (New England Biolabs) at 37°C for 2 hr. .. Prior to bisulfite conversion, fully methylated positive controls were also generated by incubating samples with S-Adenosyl methionine (SAM) and DNA methyltransferase (New England Biolabs) at 37°C for 2 hr.

    IA:

    Article Title: Curcumin Down-Regulates DNA Methyltransferase 1 and Plays an Anti-Leukemic Role in Acute Myeloid Leukemia
    Article Snippet: The primers for amplification of p15INK4B and its bisulfite-converted promoter region, and also for DNMT1 and its Sp1-binding promoter region, were purchased from Sigma-Aldrich or Integrated DNA Technology (IDT, Coralville, IA). .. M. SssI methylase, s-adenosylmethionine (SAM) (3.2 mM) and 10X incubation buffer were purchased from New England Biolabs (Ipswich, MA).

    Agarose Gel Electrophoresis:

    Article Title: SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC
    Article Snippet: Methylation was assayed by digesting an aliquot of the plasmid with Hph I, followed by agarose gel electrophoresis and Southern blotting. .. PCR products (2μg) purified using a QIAquick PCR purification kit were treated with 8 units of dam methylase and 800μM S-adenosylmethionine (New England Biolabs) at 37°C overnight.

    Plasmid Preparation:

    Article Title: MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma
    Article Snippet: Prior to bisulfite conversion, fully methylated positive controls were also generated by incubating samples with S-Adenosyl methionine (SAM) and DNA methyltransferase (New England Biolabs) at 37°C for 2 hr. .. Prior to bisulfite conversion, fully methylated positive controls were also generated by incubating samples with S-Adenosyl methionine (SAM) and DNA methyltransferase (New England Biolabs) at 37°C for 2 hr.

    Article Title: Turn-on DNA Damage Sensors for the Direct Detection of 8-Oxoguanine and Photoproducts in Native DNA
    Article Snippet: All designed DNA targets were annealed in 1× annealing buffer (10 mM Tris-HCl, pH 7.9, 150 mM NaCl, 10 mM MgCl2 , 1mM DTT) using the following procedure: heating to 95 °C for 7 min, cooling to 56 °C at a rate of 0.1 °C sec−1 , equilibrating at 56 °C for 5 min, cooling to 25 °C at a rate of 0.1 °C sec−1 and finally equilibrating at 25 °C for 10 min using a Labnet Multi Gene II thermocycler. .. A pETDuet-derived plasmid (7667 bp) was fully methylated, generating pETm, by incubation with S-adenosylmethionine and M.SssI CpG methyltransferase (New England Biolabs) according to the manufacturer’s protocol. .. The extent of protection was determined by exposure to the methylation sensitive endonuclease BstUI.

    Article Title: SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC
    Article Snippet: Methylation was assayed by digesting an aliquot of the plasmid with Hph I, followed by agarose gel electrophoresis and Southern blotting. .. PCR products (2μg) purified using a QIAquick PCR purification kit were treated with 8 units of dam methylase and 800μM S-adenosylmethionine (New England Biolabs) at 37°C overnight.

    Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation
    Article Snippet: Cloned vector was then methylated in vitro utilizing Ss1 methylases (New England Biolabs, Frankfurt, Germany) according to the manufacturer's instruction. .. Briefly, 1 μg of plasmid DNA was added to a reaction containing CpG methyltransferase (Sss1, 4 units/μl) in the presence of 160 μ m S -adenosylmethionine (New England Biolabs) and incubated for 4 h at 37 °C, and S -adenosylmethionine was replenished after every 2 h. Unmethylated control reaction contained the NPC1L1 promoter construct and methylases but not S -adenosylmethionine. .. Plasmid DNA was then purified by using Qiagen miniprep kit and quantified using a spectrophotometer.

    Article Title: Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells
    Article Snippet: To allow T/A cloning, the vector was digested with Hin dIII and Pst I (both from New England Biolabs), blunted using T4 DNA polymerase (New England Biolabs) and subsequently thymidine-tailed. .. 18 µg of pCpGL/2031 were in vitro methylated using the CpG methyltransferase M.SssI (4 units/µg), in the presence of 640 µM S-Adenosylmethionine (both from New England Biolabs) for 3 hours at 37°C and designated pCpGL/2031_M.SssI.

    Article Title: Twin-Arginine Translocation System in Helicobacter pylori: TatC, but Not TatB, Is Essential for Viability
    Article Snippet: In this plasmid, the tatC gene is under the control of a (IPTG-inducible) Ptac promoter. .. Plasmid pSLB495 was methylated in the presence of S -adenosylmethionine (New England Biolabs, Ipswich, MA) and cell-free extracts from H. pylori parental strain 43504, as described by Boneca et al. ( ). .. Methylated plasmid pSLB495 was then introduced into strain 43504, and chloramphenicol-resistant mutants were isolated.

    Article Title: CREB/ATF-dependent T cell receptor-induced FoxP3 gene expression: a role for DNA methylation
    Article Snippet: The PCR product was cloned using the TOPO TA Cloning kit (Invitrogen). .. For plasmid methylation, the +4,301 to +4,500 CpG island region was amplified by PCR, and 5 μg of PCR product was incubated with 80 U of SssI methylase and 640 μM S-adenosylmethionine (New England Biolabs) for 2 h at 37°C. .. DNA methylation was verified using AatII, which is a methylation-sensitive restriction enzyme.

    Article Title: Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule
    Article Snippet: The p39E plasmid and its promoter deletion derivatives ( ) are schematically depicted in Figure A. .. Plasmids were incubated with 5-fold molar excess of TFO−C141S, M.SssI, C141S, or TFO in a buffer containing 20 mM Tris, 50 mM NaCl, 10 mM MgCl2 , pH 7.9, with or without 640 μM S -adenosylmethionine (SAM) (New England Biolabs, Ipswich, MA) at 30 °C.

    Article Title: Modulation of Dnmt3b function in vitro by interactions with Dnmt3L, Dnmt3a and Dnmt3b splice variants
    Article Snippet: Films with pull down and input were scanned and the composite images shown generated without further computerized image adjustment (e.g. alterations in contrast, brightness, etc.). .. Activity assay conditions, adapted from Takeshima et al . , were 20 mM Tris (pH 7.4), 5 mM EDTA, 25 mM NaCl, 10% glycerol, 200 µM DTT, 80 µM S -adenosyl-l -methionine (SAM, New England Biolabs) and 1.5 µg pSL301 plasmid containing the SAT2 sequence. .. The reaction mixture was incubated at 37°C for 7 h, with fresh SAM added after the first 4 h. For reactions comparing enzyme activity with and without Dnmt3L, 0.15 nmol of each purified methyltransferase was mixed in equimolar concentrations.

    Article Title: Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation
    Article Snippet: Finally, the biotin-DNA was detected by chemiluminescence and visualized by Biospectrum® 510 Imaging System (Upland, CA, USA). .. The methylated plasmids (Met-pGL3-446/-396) were generated by incubating 1 μg of plasmid DNA (pGL3 -446/-396) with 10 units Hpall methyltransferase in 10 μl 1 × Hpall methyltransferase buffer and 80 μM S-adenosylmethionine according to the manufacturer’s protocols (New England Biolabs, Inc.). .. Reactions were carried out at 37 °C 1 h followed by 15 min at 65 °C to inactivate the methylase, purified by polyacrylamide gel electrophoresis.

    Software:

    Article Title: SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC
    Article Snippet: Blots were hybridized with a radiolabeled oriC probe (248 bp Hind III and Pst I fragment obtained from pOC170), scanned with a BioRad Personal Molecular Imager FX and images analyzed using Quantity One (BioRad) software. .. PCR products (2μg) purified using a QIAquick PCR purification kit were treated with 8 units of dam methylase and 800μM S-adenosylmethionine (New England Biolabs) at 37°C overnight.

    Negative Control:

    Article Title: Modulation of Dnmt3b function in vitro by interactions with Dnmt3L, Dnmt3a and Dnmt3b splice variants
    Article Snippet: Activity assay conditions, adapted from Takeshima et al . , were 20 mM Tris (pH 7.4), 5 mM EDTA, 25 mM NaCl, 10% glycerol, 200 µM DTT, 80 µM S -adenosyl-l -methionine (SAM, New England Biolabs) and 1.5 µg pSL301 plasmid containing the SAT2 sequence. .. For reactions combining Dnmt3b’s with Dnmt3a and Dnmt3L, 0.06 nmol of each purified methyltransferase was used.

    Sample Prep:

    Article Title: Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping
    Article Snippet: Paragraph title: AFM sample preparation ... Protein samples were diluted to their final concentration in 1× buffer solution (10 mM MgCl2 , 10 mM KCl, 10 mM Tris–HCl pH 7.9) in the presence of 100 μM S -adenosyl-l -methionine (AdoMet, New England Biolabs, MA, USA).

    In Vitro:

    Article Title: Dietary Flavones as Dual Inhibitors of DNA Methyltransferases and Histone Methyltransferases
    Article Snippet: Paragraph title: In vitro promoter methylation and digestion with the methylation- sensitive HpaII enzyme ... The methylation reactions were carried out in 1X M.SssI buffer with 160 mM SAM S-adenosylmethionine (supplied with M.SssI by New England Biolabs).

    Article Title: Turn-on DNA Damage Sensors for the Direct Detection of 8-Oxoguanine and Photoproducts in Native DNA
    Article Snippet: Paragraph title: Generation of DNA oxidation IN VITRO ... A pETDuet-derived plasmid (7667 bp) was fully methylated, generating pETm, by incubation with S-adenosylmethionine and M.SssI CpG methyltransferase (New England Biolabs) according to the manufacturer’s protocol.

    Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation
    Article Snippet: Paragraph title: Cloning and in Vitro Methylation of pCpG Free-L1 Vector ... Briefly, 1 μg of plasmid DNA was added to a reaction containing CpG methyltransferase (Sss1, 4 units/μl) in the presence of 160 μ m S -adenosylmethionine (New England Biolabs) and incubated for 4 h at 37 °C, and S -adenosylmethionine was replenished after every 2 h. Unmethylated control reaction contained the NPC1L1 promoter construct and methylases but not S -adenosylmethionine.

    Article Title: Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells
    Article Snippet: The pCpGL-basic with the cloned upstream region was designated pCpGL/2031. .. 18 µg of pCpGL/2031 were in vitro methylated using the CpG methyltransferase M.SssI (4 units/µg), in the presence of 640 µM S-Adenosylmethionine (both from New England Biolabs) for 3 hours at 37°C and designated pCpGL/2031_M.SssI. .. The unmethylated control reporter vector (pCpGL/2031_mock) was treated as above but without methyltransferase.

    Article Title: Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation
    Article Snippet: Paragraph title: In vitro DNA methylation and transient transfection ... The methylated plasmids (Met-pGL3-446/-396) were generated by incubating 1 μg of plasmid DNA (pGL3 -446/-396) with 10 units Hpall methyltransferase in 10 μl 1 × Hpall methyltransferase buffer and 80 μM S-adenosylmethionine according to the manufacturer’s protocols (New England Biolabs, Inc.).

    DNA Methylation Assay:

    Article Title: Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells
    Article Snippet: Paragraph title: Cloning of the upstream region of precursor mir-142 , construction of expression constructs, in vitro DNA methylation and luciferase experiments ... 18 µg of pCpGL/2031 were in vitro methylated using the CpG methyltransferase M.SssI (4 units/µg), in the presence of 640 µM S-Adenosylmethionine (both from New England Biolabs) for 3 hours at 37°C and designated pCpGL/2031_M.SssI.

    Article Title: Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation
    Article Snippet: Paragraph title: In vitro DNA methylation and transient transfection ... The methylated plasmids (Met-pGL3-446/-396) were generated by incubating 1 μg of plasmid DNA (pGL3 -446/-396) with 10 units Hpall methyltransferase in 10 μl 1 × Hpall methyltransferase buffer and 80 μM S-adenosylmethionine according to the manufacturer’s protocols (New England Biolabs, Inc.).

    Concentration Assay:

    Article Title: Synthesis of 5? cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N7)-methyl transferase
    Article Snippet: Betaplate Scint (Wallac) scintillation fluid was added, and the methylation of RNA substrates was measured in counts per minute (c.p.m.) using a Wallac 1450 MicroBeta TriLux Liquid Scintillation Counter. .. Methylation of purified Gppp-RNAs ( 2–4 ), ( 9–10 ) (∼70 nanomol) to give 7m GpppN-RNAs ( 12–15 ) and 7m GpppN2′-Om -RNA ( 16 ) was carried out using 0.25 μM N 7 -hMTase and 0.4 mM S -adenosylmethionine (New England Biolabs) in 40 mM Tris-HCl, pH 8, with 5 mM dithiothreitol in a 1725-μL reaction volume (Gppp-RNAs at a final concentration of 40 μM) at 30°C. .. N 7 -methylation was monitored using IEX-HPLC analysis of an aliquot from the reaction mixture and passed through ZipTip C18 (see procedure below) prior analysis to remove protein.

    Article Title: Turn-on DNA Damage Sensors for the Direct Detection of 8-Oxoguanine and Photoproducts in Native DNA
    Article Snippet: A pETDuet-derived plasmid (7667 bp) was fully methylated, generating pETm, by incubation with S-adenosylmethionine and M.SssI CpG methyltransferase (New England Biolabs) according to the manufacturer’s protocol. .. To introduce oxidation, 708 nM annealed oligonucleotide, 8 ng/μL pETm, or 50 ng/μL HeLa DNA was treated with 1 mM H2 O2 in the presence of 30 μM CuCl2 for 30 min at RT, followed by quenching with 1 mM EDTA.

    Article Title: CREB/ATF-dependent T cell receptor-induced FoxP3 gene expression: a role for DNA methylation
    Article Snippet: For plasmid methylation, the +4,301 to +4,500 CpG island region was amplified by PCR, and 5 μg of PCR product was incubated with 80 U of SssI methylase and 640 μM S-adenosylmethionine (New England Biolabs) for 2 h at 37°C. .. DNA methylation was verified using AatII, which is a methylation-sensitive restriction enzyme.

    Article Title: Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping
    Article Snippet: Immediately prior to protein or DNA deposition, the top layer of the mica was cleaved using Scotch tape to reveal an atomically flat surface. .. Protein samples were diluted to their final concentration in 1× buffer solution (10 mM MgCl2 , 10 mM KCl, 10 mM Tris–HCl pH 7.9) in the presence of 100 μM S -adenosyl-l -methionine (AdoMet, New England Biolabs, MA, USA). .. Droplets of 20 μl were deposited onto freshly cleaved mica discs and incubated at room temperature for 2 min. After incubation, excess protein was rinsed off with MilliQ water (Millipore System, MA, USA), excess water was wicked from the surface using tissue paper and the discs were dried under a stream of nitrogen gas.

    Staining:

    Article Title: The Drosophila G9a gene encodes a multi-catalytic histone methyltransferase required for normal development
    Article Snippet: Methylation reactions were carried out as described above with 0.5 µg of histone H3 or H4 and 250 µM of S-Adenosylmethionine (New England BioLabs). .. The reaction was stopped by addition of SDS–PAGE loading buffer and the histones were separated by 15% SDS–PAGE.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs s adenosylmethionine sam 32mm
    Effect of <t>S-adenosylmethionine</t> <t>(SAM)</t> on breast cancer cell proliferation, migration, invasion, anchorage-independent growth, and apoptosis in vitro (A) Schematic diagram of the treatment strategy for all the in vitro experiments. Human breast cancer cells MDA-MB-231 and Hs578T were treated with SAM (100 and 200 μM) by directly adding it to regular growth medium every other day from day 2 until they were harvested. (B) Human breast cancer cells MDA-MB-231 and Hs578T were plated in 6-well plates and treated with vehicle alone as control or SAM (100 and 200 μM). Cell growth rate in each group was determined on day 1, 3, 5, and 7 by Coulter counter as described in Methods. Results are shown as bar graphs of data obtained from three different experiments. (C) Wound healing assay for determining the migration capacity of the cells was carried out by making a cross-like scratch on the plate when they reached 90% confluency. Control and SAM (100 and 200 μM) treated cells were grown in culture media containing 2% FBS and migrating cells were photographed and recorded at different time points, and percentage of wound healing with respect to initial scratch (T0) was calculated using the equation described in ‘Supplementary Materials’. The results are represented as bar graphs obtained from three experiments. (D) Boyden chamber Matrigel invasion assay was used to measure the invasiveness of control and SAM-treated (100 and 200 μM) MDA-MB-231 and Hs578T cells. The cells were placed in the upper chamber, and conditioned media used as ‘chemoattractant’ was added into the lower chamber. Following an incubation period of 18 hours, the invasion process was stopped and the invaded cells from control and 100 and 200 μM SAM-treated groups were fixed, stained and randomly selected fields were counted under the microscope and averaged. Representative image of one randomly selected field for each treatment for both cell lines along with the number of cells invaded per field are shown. (E) After the usual treatment regimen, 5 × 10 3 cell from control and SAM-treated (100 μM and 200 μM) groups were plated onto soft agar for anchorage-independent growth assay. The culture media was replenished every other day for two weeks, and the number of colonies was counted. (F) Apoptosis was determined by flow cytometry after staining the control and SAM-treated cells with Annexin V/propidium iodide. Representative contour plots of annexinV-FITC staining of apoptotic cells vs. PI staining for both control and SAM-treated (100 μM) cells are shown. The bar graphs on the right panels show the total percentages of apoptotic cells for different treatments. Results are presented as the mean ± SEM from control and SAM-treated experimental cells. Significant differences were determined using ANOVA followed by post hoc Bonferroni test and are represented by asterisks ( * P
    S Adenosylmethionine Sam 32mm, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/s adenosylmethionine sam 32mm/product/New England Biolabs
    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    s adenosylmethionine sam 32mm - by Bioz Stars, 2019-10
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs h s adenosylmethionine
    Functional characterization of the hyperreactive cysteines in PRMT1 a , Crystal structure of rat PRMT1 26 (green, PDB: 1ORI) showing the hyperreactive cysteine C101 in contact with an S-adenosylhomocysteine (SAH) cofactor (cyan). b , Wild-type and C101A mutant of human PRMT1 were labeled with the IA-probe, followed by click-chemistry to incorporate a fluorescent rhodamine tag. In-gel fluorescence demonstrates robust labeling of the wild-type (WT) but not C101A mutant PRMT1 and IA-labeling of WT PRMT1 is inhibited by HNE. c , Catalytic activity of purified WT, but not C101A PRMT1 is inhibited by HNE as measured by monitoring transfer of 3 H-methyl from 3 <t>H-S-adenosylmethionine</t> (SAM) to a histone 4 substrate.
    H S Adenosylmethionine, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/h s adenosylmethionine/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    h s adenosylmethionine - by Bioz Stars, 2019-10
    99/100 stars
      Buy from Supplier

    92
    New England Biolabs non radioactive adomet
    Gene structure and targeting strategy for the endothelin converting enzyme 2 ( ECE2 ) /EEF1AKMT4 locus. ( A ) Topology diagram of the 7BS MTase fold. α-helices and β-strands are depicted as grey boxes (denoted Z and A–E) and black arrows (denoted 1–7), respectively, and annotated according to previously established nomenclature ( 50 ). ( B ) Organization and annotation of the human ECE2 locus. Top, current annotation of the ECE2 locus. Regions in the genomic DNA corresponding to annotated exons of ECE2 (located on the forward stand) and CAMK2N2 (located on the reverse strand) are indicated in blue and green, respectively (based on the annotated gene structure according to Ensembl (assembly GRCh37)). Middle, tracks representing spliced expressed sequence tags (ESTs) exported from the UCSC genome browser. Bottom, alternative gene model supported by the EST data. ( C ) Gene targeting strategy and disruption of the EEF1AKMT4 locus by CRISPR/Cas9 technology. Top, schematic representation of the EEF1AKMT4 gene. Exons and introns are represented by boxes (gray, coding region; white, untranslated regions) and lines, respectively. An arrow indicates the region targeted by CRISPR. Bottom, DNA and protein sequence of targeted region of EEF1AKMT4 locus in HAP-1 wild-type (WT) and EEF1AKMT4 knockout (KO) cells. The conserved motif Post I is indicated by a rectangle and the last residue of the motif, D88, is shown in red. ( D ) Structural support for an involvement of D88 in coordinating <t>AdoMet</t> binding. The figure is based on a previously published structure (PDB ID: 2PXX) of <t>eEF1A-KMT4</t> in complex with S -adenosylhomocysteine (AdoHcy; the unmethylated counterpart of AdoMet) ( 32 ). AdoHcy and D88 are represented as sticks and the second β-strand (β2) is represented as a cartoon. Possible hydrogen bonds between the carboxyl group of D88 and the ribose moiety of AdoHcy are shown (dashed lines). The figure was generated using the PyMOL Molecular Graphics System, Version 1.3 (Schrodinger, LLC).
    Non Radioactive Adomet, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/non radioactive adomet/product/New England Biolabs
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    non radioactive adomet - by Bioz Stars, 2019-10
    92/100 stars
      Buy from Supplier

    Image Search Results


    Effect of S-adenosylmethionine (SAM) on breast cancer cell proliferation, migration, invasion, anchorage-independent growth, and apoptosis in vitro (A) Schematic diagram of the treatment strategy for all the in vitro experiments. Human breast cancer cells MDA-MB-231 and Hs578T were treated with SAM (100 and 200 μM) by directly adding it to regular growth medium every other day from day 2 until they were harvested. (B) Human breast cancer cells MDA-MB-231 and Hs578T were plated in 6-well plates and treated with vehicle alone as control or SAM (100 and 200 μM). Cell growth rate in each group was determined on day 1, 3, 5, and 7 by Coulter counter as described in Methods. Results are shown as bar graphs of data obtained from three different experiments. (C) Wound healing assay for determining the migration capacity of the cells was carried out by making a cross-like scratch on the plate when they reached 90% confluency. Control and SAM (100 and 200 μM) treated cells were grown in culture media containing 2% FBS and migrating cells were photographed and recorded at different time points, and percentage of wound healing with respect to initial scratch (T0) was calculated using the equation described in ‘Supplementary Materials’. The results are represented as bar graphs obtained from three experiments. (D) Boyden chamber Matrigel invasion assay was used to measure the invasiveness of control and SAM-treated (100 and 200 μM) MDA-MB-231 and Hs578T cells. The cells were placed in the upper chamber, and conditioned media used as ‘chemoattractant’ was added into the lower chamber. Following an incubation period of 18 hours, the invasion process was stopped and the invaded cells from control and 100 and 200 μM SAM-treated groups were fixed, stained and randomly selected fields were counted under the microscope and averaged. Representative image of one randomly selected field for each treatment for both cell lines along with the number of cells invaded per field are shown. (E) After the usual treatment regimen, 5 × 10 3 cell from control and SAM-treated (100 μM and 200 μM) groups were plated onto soft agar for anchorage-independent growth assay. The culture media was replenished every other day for two weeks, and the number of colonies was counted. (F) Apoptosis was determined by flow cytometry after staining the control and SAM-treated cells with Annexin V/propidium iodide. Representative contour plots of annexinV-FITC staining of apoptotic cells vs. PI staining for both control and SAM-treated (100 μM) cells are shown. The bar graphs on the right panels show the total percentages of apoptotic cells for different treatments. Results are presented as the mean ± SEM from control and SAM-treated experimental cells. Significant differences were determined using ANOVA followed by post hoc Bonferroni test and are represented by asterisks ( * P

    Journal: Oncotarget

    Article Title: Methyl donor S-adenosylmethionine (SAM) supplementation attenuates breast cancer growth, invasion, and metastasis in vivo; therapeutic and chemopreventive applications

    doi: 10.18632/oncotarget.23704

    Figure Lengend Snippet: Effect of S-adenosylmethionine (SAM) on breast cancer cell proliferation, migration, invasion, anchorage-independent growth, and apoptosis in vitro (A) Schematic diagram of the treatment strategy for all the in vitro experiments. Human breast cancer cells MDA-MB-231 and Hs578T were treated with SAM (100 and 200 μM) by directly adding it to regular growth medium every other day from day 2 until they were harvested. (B) Human breast cancer cells MDA-MB-231 and Hs578T were plated in 6-well plates and treated with vehicle alone as control or SAM (100 and 200 μM). Cell growth rate in each group was determined on day 1, 3, 5, and 7 by Coulter counter as described in Methods. Results are shown as bar graphs of data obtained from three different experiments. (C) Wound healing assay for determining the migration capacity of the cells was carried out by making a cross-like scratch on the plate when they reached 90% confluency. Control and SAM (100 and 200 μM) treated cells were grown in culture media containing 2% FBS and migrating cells were photographed and recorded at different time points, and percentage of wound healing with respect to initial scratch (T0) was calculated using the equation described in ‘Supplementary Materials’. The results are represented as bar graphs obtained from three experiments. (D) Boyden chamber Matrigel invasion assay was used to measure the invasiveness of control and SAM-treated (100 and 200 μM) MDA-MB-231 and Hs578T cells. The cells were placed in the upper chamber, and conditioned media used as ‘chemoattractant’ was added into the lower chamber. Following an incubation period of 18 hours, the invasion process was stopped and the invaded cells from control and 100 and 200 μM SAM-treated groups were fixed, stained and randomly selected fields were counted under the microscope and averaged. Representative image of one randomly selected field for each treatment for both cell lines along with the number of cells invaded per field are shown. (E) After the usual treatment regimen, 5 × 10 3 cell from control and SAM-treated (100 μM and 200 μM) groups were plated onto soft agar for anchorage-independent growth assay. The culture media was replenished every other day for two weeks, and the number of colonies was counted. (F) Apoptosis was determined by flow cytometry after staining the control and SAM-treated cells with Annexin V/propidium iodide. Representative contour plots of annexinV-FITC staining of apoptotic cells vs. PI staining for both control and SAM-treated (100 μM) cells are shown. The bar graphs on the right panels show the total percentages of apoptotic cells for different treatments. Results are presented as the mean ± SEM from control and SAM-treated experimental cells. Significant differences were determined using ANOVA followed by post hoc Bonferroni test and are represented by asterisks ( * P

    Article Snippet: Cells were treated with SAM (New England Biolabs, Mississauga, Ontario, Canada; Catalog # B9003S) by directly adding it to regular growth medium under sterile conditions following the treatment plan shown in Figure .

    Techniques: Migration, In Vitro, Multiple Displacement Amplification, Wound Healing Assay, Invasion Assay, Incubation, Staining, Microscopy, Growth Assay, Flow Cytometry, Cytometry

    Functional characterization of the hyperreactive cysteines in PRMT1 a , Crystal structure of rat PRMT1 26 (green, PDB: 1ORI) showing the hyperreactive cysteine C101 in contact with an S-adenosylhomocysteine (SAH) cofactor (cyan). b , Wild-type and C101A mutant of human PRMT1 were labeled with the IA-probe, followed by click-chemistry to incorporate a fluorescent rhodamine tag. In-gel fluorescence demonstrates robust labeling of the wild-type (WT) but not C101A mutant PRMT1 and IA-labeling of WT PRMT1 is inhibited by HNE. c , Catalytic activity of purified WT, but not C101A PRMT1 is inhibited by HNE as measured by monitoring transfer of 3 H-methyl from 3 H-S-adenosylmethionine (SAM) to a histone 4 substrate.

    Journal: Nature

    Article Title: Quantitative reactivity profiling predicts functional cysteines in proteomes

    doi: 10.1038/nature09472

    Figure Lengend Snippet: Functional characterization of the hyperreactive cysteines in PRMT1 a , Crystal structure of rat PRMT1 26 (green, PDB: 1ORI) showing the hyperreactive cysteine C101 in contact with an S-adenosylhomocysteine (SAH) cofactor (cyan). b , Wild-type and C101A mutant of human PRMT1 were labeled with the IA-probe, followed by click-chemistry to incorporate a fluorescent rhodamine tag. In-gel fluorescence demonstrates robust labeling of the wild-type (WT) but not C101A mutant PRMT1 and IA-labeling of WT PRMT1 is inhibited by HNE. c , Catalytic activity of purified WT, but not C101A PRMT1 is inhibited by HNE as measured by monitoring transfer of 3 H-methyl from 3 H-S-adenosylmethionine (SAM) to a histone 4 substrate.

    Article Snippet: 500 ng of recombinant human PRMT1 (wild-type or C101A mutant) was pre-incubated with 4-hydroxy-2-nonenal (HNE, Calbiochem) for 30 minutes and methylation activity was monitored after addition of 1 mg of recombinant histone 4 (M2504S; NEB) and 3 H-S-adenosylmethionine (SAM, 2 μCi) in methylation buffer (20 mM Tris [pH 8.0], 200mM NaCl, 0.4 mM EDTA).

    Techniques: Functional Assay, Mutagenesis, Labeling, IA, Fluorescence, Activity Assay, Purification

    Gene structure and targeting strategy for the endothelin converting enzyme 2 ( ECE2 ) /EEF1AKMT4 locus. ( A ) Topology diagram of the 7BS MTase fold. α-helices and β-strands are depicted as grey boxes (denoted Z and A–E) and black arrows (denoted 1–7), respectively, and annotated according to previously established nomenclature ( 50 ). ( B ) Organization and annotation of the human ECE2 locus. Top, current annotation of the ECE2 locus. Regions in the genomic DNA corresponding to annotated exons of ECE2 (located on the forward stand) and CAMK2N2 (located on the reverse strand) are indicated in blue and green, respectively (based on the annotated gene structure according to Ensembl (assembly GRCh37)). Middle, tracks representing spliced expressed sequence tags (ESTs) exported from the UCSC genome browser. Bottom, alternative gene model supported by the EST data. ( C ) Gene targeting strategy and disruption of the EEF1AKMT4 locus by CRISPR/Cas9 technology. Top, schematic representation of the EEF1AKMT4 gene. Exons and introns are represented by boxes (gray, coding region; white, untranslated regions) and lines, respectively. An arrow indicates the region targeted by CRISPR. Bottom, DNA and protein sequence of targeted region of EEF1AKMT4 locus in HAP-1 wild-type (WT) and EEF1AKMT4 knockout (KO) cells. The conserved motif Post I is indicated by a rectangle and the last residue of the motif, D88, is shown in red. ( D ) Structural support for an involvement of D88 in coordinating AdoMet binding. The figure is based on a previously published structure (PDB ID: 2PXX) of eEF1A-KMT4 in complex with S -adenosylhomocysteine (AdoHcy; the unmethylated counterpart of AdoMet) ( 32 ). AdoHcy and D88 are represented as sticks and the second β-strand (β2) is represented as a cartoon. Possible hydrogen bonds between the carboxyl group of D88 and the ribose moiety of AdoHcy are shown (dashed lines). The figure was generated using the PyMOL Molecular Graphics System, Version 1.3 (Schrodinger, LLC).

    Journal: Nucleic Acids Research

    Article Title: Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

    doi: 10.1093/nar/gkx432

    Figure Lengend Snippet: Gene structure and targeting strategy for the endothelin converting enzyme 2 ( ECE2 ) /EEF1AKMT4 locus. ( A ) Topology diagram of the 7BS MTase fold. α-helices and β-strands are depicted as grey boxes (denoted Z and A–E) and black arrows (denoted 1–7), respectively, and annotated according to previously established nomenclature ( 50 ). ( B ) Organization and annotation of the human ECE2 locus. Top, current annotation of the ECE2 locus. Regions in the genomic DNA corresponding to annotated exons of ECE2 (located on the forward stand) and CAMK2N2 (located on the reverse strand) are indicated in blue and green, respectively (based on the annotated gene structure according to Ensembl (assembly GRCh37)). Middle, tracks representing spliced expressed sequence tags (ESTs) exported from the UCSC genome browser. Bottom, alternative gene model supported by the EST data. ( C ) Gene targeting strategy and disruption of the EEF1AKMT4 locus by CRISPR/Cas9 technology. Top, schematic representation of the EEF1AKMT4 gene. Exons and introns are represented by boxes (gray, coding region; white, untranslated regions) and lines, respectively. An arrow indicates the region targeted by CRISPR. Bottom, DNA and protein sequence of targeted region of EEF1AKMT4 locus in HAP-1 wild-type (WT) and EEF1AKMT4 knockout (KO) cells. The conserved motif Post I is indicated by a rectangle and the last residue of the motif, D88, is shown in red. ( D ) Structural support for an involvement of D88 in coordinating AdoMet binding. The figure is based on a previously published structure (PDB ID: 2PXX) of eEF1A-KMT4 in complex with S -adenosylhomocysteine (AdoHcy; the unmethylated counterpart of AdoMet) ( 32 ). AdoHcy and D88 are represented as sticks and the second β-strand (β2) is represented as a cartoon. Possible hydrogen bonds between the carboxyl group of D88 and the ribose moiety of AdoHcy are shown (dashed lines). The figure was generated using the PyMOL Molecular Graphics System, Version 1.3 (Schrodinger, LLC).

    Article Snippet: To identify the residue(s) targeted by eEF1A-KMT4, recombinant eEF1A1 was treated with eEF1A-KMT4 and non-radioactive AdoMet, followed by proteolytic digestion with chymotrypsin and the resulting peptides were analyzed by MS.

    Techniques: Sequencing, CRISPR, Knock-Out, Gene Knockout, Binding Assay, Generated

    eEF1A-KMT4-mediated methylation of K36 in eEF1A proteins in vitro . ( A ) Nucleotide-dependent methylation of recombinant eEF1A1 by eEF1A-KMT4. Recombinant eEF1A1 was incubated with [ 3 H]-AdoMet and eEF1A-KMT4, as well as with adenosinetriphosphate (ATP) or GTP as indicated. Top, Ponceau S stained membrane of proteins. Bottom, visualization of methylation by fluorography of membrane in upper panel. ( B ) Comparison of eEF1A1 and eEF1A2 as substrates for eEF1A-KMT4-mediated methylation. Recombinant eEF1A1 or eEF1A2 was incubated with varying amounts of eEF1A-KMT4. Methylation was quantified as trichloroacetic acid (TCA)-insoluble radioactivity. Error bars represent the standard deviation ( n = 3). ( C ) Evaluation of eEF1A point mutants as substrates for eEF1A-KMT4-mediated methylation. WT eEF1A1 and eEF1A2 or the corresponding Lys36→Arg (K36R) mutant proteins were incubated with recombinant eEF1A-KMT4 in the presence of [ 3 H]-AdoMet. Reactions were analyzed as in (A). Note: In panels (A) and (C), automethylation of recombinant eEF1A-KMT4 is observed, but such automethylation was not observed in the experiments shown in Figure 2A , B and D . This apparent discrepancy is likely because less enzyme and of lower specific activity, was used in Figure 2 .

    Journal: Nucleic Acids Research

    Article Title: Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

    doi: 10.1093/nar/gkx432

    Figure Lengend Snippet: eEF1A-KMT4-mediated methylation of K36 in eEF1A proteins in vitro . ( A ) Nucleotide-dependent methylation of recombinant eEF1A1 by eEF1A-KMT4. Recombinant eEF1A1 was incubated with [ 3 H]-AdoMet and eEF1A-KMT4, as well as with adenosinetriphosphate (ATP) or GTP as indicated. Top, Ponceau S stained membrane of proteins. Bottom, visualization of methylation by fluorography of membrane in upper panel. ( B ) Comparison of eEF1A1 and eEF1A2 as substrates for eEF1A-KMT4-mediated methylation. Recombinant eEF1A1 or eEF1A2 was incubated with varying amounts of eEF1A-KMT4. Methylation was quantified as trichloroacetic acid (TCA)-insoluble radioactivity. Error bars represent the standard deviation ( n = 3). ( C ) Evaluation of eEF1A point mutants as substrates for eEF1A-KMT4-mediated methylation. WT eEF1A1 and eEF1A2 or the corresponding Lys36→Arg (K36R) mutant proteins were incubated with recombinant eEF1A-KMT4 in the presence of [ 3 H]-AdoMet. Reactions were analyzed as in (A). Note: In panels (A) and (C), automethylation of recombinant eEF1A-KMT4 is observed, but such automethylation was not observed in the experiments shown in Figure 2A , B and D . This apparent discrepancy is likely because less enzyme and of lower specific activity, was used in Figure 2 .

    Article Snippet: To identify the residue(s) targeted by eEF1A-KMT4, recombinant eEF1A1 was treated with eEF1A-KMT4 and non-radioactive AdoMet, followed by proteolytic digestion with chymotrypsin and the resulting peptides were analyzed by MS.

    Techniques: Methylation, In Vitro, Recombinant, Incubation, Staining, Radioactivity, Standard Deviation, Mutagenesis, Activity Assay

    Identification of eEF1A proteins as candidate substrates for eEF1A-KMT4. ( A ) eEF1A-KMT4-mediated methylation in protein extracts. Protein extracts from HAP-1 wild-type (WT) or EEF1AKMT4 KO cells were incubated with [ 3 H]-AdoMet and with recombinant eEF1A-KMT4 as indicated. Top panel, Ponceau S-stained membrane of sodium dodecyl sulphate (SDS-PAGE)-separated methylation reactions. Bottom, visualization of methylation by fluorography of membrane in upper panel. ( B ) Mutation of D88 abrogates the enzymatic activity of eEF1A-KMT4. Protein extract from HAP-1 EEF1AKMT4 KO cells incubated with [ 3 H]-AdoMet in the presence of either recombinant WT eEF1A-KMT4 or the corresponding D88A mutant protein. Reactions were analyzed as in (A). ( C ) Outline of chromatography based enrichment strategy for eEF1A-KMT4 substrates. In vitro methylated protein extract was loaded onto an anion exchange (Q) column and, in turn, the flow-through (FTQ) was loaded onto a cation exchange (S) column. Proteins were eluted from both columns with a step gradient of increasing NaCl. ( D ) Enrichment of the ∼50 kDa eEF1A-KMT4 substrate. Proteins were eluted from ion exchange columns with a step gradient ramped from 150 mM (0.15) to 1 M (1.0) NaCl according to the scheme depicted in (C). Collected fractions were analyzed by fluorography as in (A). ( E ) SDS-PAGE analysis of the main substrate-enriched fraction (0.3S). The fraction containing the bulk of methylated substrate in (D) eluted at 300 mM NaCl from the S column (0.3S). The most prominent proteins in the major ∼50 kDaband were identified as eEF1A1 and eEF1A2 by mass spectrometry (MS) (See Table 1 ).

    Journal: Nucleic Acids Research

    Article Title: Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

    doi: 10.1093/nar/gkx432

    Figure Lengend Snippet: Identification of eEF1A proteins as candidate substrates for eEF1A-KMT4. ( A ) eEF1A-KMT4-mediated methylation in protein extracts. Protein extracts from HAP-1 wild-type (WT) or EEF1AKMT4 KO cells were incubated with [ 3 H]-AdoMet and with recombinant eEF1A-KMT4 as indicated. Top panel, Ponceau S-stained membrane of sodium dodecyl sulphate (SDS-PAGE)-separated methylation reactions. Bottom, visualization of methylation by fluorography of membrane in upper panel. ( B ) Mutation of D88 abrogates the enzymatic activity of eEF1A-KMT4. Protein extract from HAP-1 EEF1AKMT4 KO cells incubated with [ 3 H]-AdoMet in the presence of either recombinant WT eEF1A-KMT4 or the corresponding D88A mutant protein. Reactions were analyzed as in (A). ( C ) Outline of chromatography based enrichment strategy for eEF1A-KMT4 substrates. In vitro methylated protein extract was loaded onto an anion exchange (Q) column and, in turn, the flow-through (FTQ) was loaded onto a cation exchange (S) column. Proteins were eluted from both columns with a step gradient of increasing NaCl. ( D ) Enrichment of the ∼50 kDa eEF1A-KMT4 substrate. Proteins were eluted from ion exchange columns with a step gradient ramped from 150 mM (0.15) to 1 M (1.0) NaCl according to the scheme depicted in (C). Collected fractions were analyzed by fluorography as in (A). ( E ) SDS-PAGE analysis of the main substrate-enriched fraction (0.3S). The fraction containing the bulk of methylated substrate in (D) eluted at 300 mM NaCl from the S column (0.3S). The most prominent proteins in the major ∼50 kDaband were identified as eEF1A1 and eEF1A2 by mass spectrometry (MS) (See Table 1 ).

    Article Snippet: To identify the residue(s) targeted by eEF1A-KMT4, recombinant eEF1A1 was treated with eEF1A-KMT4 and non-radioactive AdoMet, followed by proteolytic digestion with chymotrypsin and the resulting peptides were analyzed by MS.

    Techniques: Methylation, Gene Knockout, Incubation, Recombinant, Staining, SDS Page, Mutagenesis, Activity Assay, Chromatography, In Vitro, Flow Cytometry, Mass Spectrometry