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  • 95
    New England Biolabs s adenosylmethionine
    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 <t>-adenosylmethionine.</t> 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.
    S Adenosylmethionine, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 697 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    New England Biolabs m adomet
    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 <t>-adenosylmethionine.</t> 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.
    M Adomet, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 80/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs non radioactive adomet
    Identification of METTL13 as an eEF1A-specific methyltransferase. a Workflow of mass spectrometry-based quantitative peptide pull-down screen. Synthetic peptides corresponding N-terminally trimethylated (Nt-Me3) and unmethylated (Nt-Me0) eEF1A were used as baits to enrich proteins from HAP-1 cell extracts. b . c Domain organization of METTL13. The boundaries for used constructs encompassing the N-terminal (MT13-N) and the C-terminal (MT13-C) methyltransferase domains are indicated. d , e Evaluation of METTL13 constructs for eEF1A-specific methyltransferase activity. MT13-N ( d ) and MT13-C ( e ) were incubated with [ 3 <t>H]-AdoMet</t> and eEF1A1 carrying an N-terminal or C-terminal His-tag in the absence of cofactors and in the presence of either GDP or GTP. Methylation was visualized by fluorography (top panels) and the membranes were stained with Ponceau S (bottom panels) to assess protein loading
    Non Radioactive Adomet, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    New England Biolabs methyl donor adomet
    Identification of METTL13 as an eEF1A-specific methyltransferase. a Workflow of mass spectrometry-based quantitative peptide pull-down screen. Synthetic peptides corresponding N-terminally trimethylated (Nt-Me3) and unmethylated (Nt-Me0) eEF1A were used as baits to enrich proteins from HAP-1 cell extracts. b . c Domain organization of METTL13. The boundaries for used constructs encompassing the N-terminal (MT13-N) and the C-terminal (MT13-C) methyltransferase domains are indicated. d , e Evaluation of METTL13 constructs for eEF1A-specific methyltransferase activity. MT13-N ( d ) and MT13-C ( e ) were incubated with [ 3 <t>H]-AdoMet</t> and eEF1A1 carrying an N-terminal or C-terminal His-tag in the absence of cofactors and in the presence of either GDP or GTP. Methylation was visualized by fluorography (top panels) and the membranes were stained with Ponceau S (bottom panels) to assess protein loading
    Methyl Donor Adomet, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs gpc methyltransferase m cvipi
    Identification of METTL13 as an eEF1A-specific methyltransferase. a Workflow of mass spectrometry-based quantitative peptide pull-down screen. Synthetic peptides corresponding N-terminally trimethylated (Nt-Me3) and unmethylated (Nt-Me0) eEF1A were used as baits to enrich proteins from HAP-1 cell extracts. b . c Domain organization of METTL13. The boundaries for used constructs encompassing the N-terminal (MT13-N) and the C-terminal (MT13-C) methyltransferase domains are indicated. d , e Evaluation of METTL13 constructs for eEF1A-specific methyltransferase activity. MT13-N ( d ) and MT13-C ( e ) were incubated with [ 3 <t>H]-AdoMet</t> and eEF1A1 carrying an N-terminal or C-terminal His-tag in the absence of cofactors and in the presence of either GDP or GTP. Methylation was visualized by fluorography (top panels) and the membranes were stained with Ponceau S (bottom panels) to assess protein loading
    Gpc Methyltransferase M Cvipi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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.

    Journal: Journal of Bacteriology

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

    doi: 10.1128/JB.00108-07

    Figure Lengend 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.

    Article Snippet: 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.

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

    Human METTL12 is a protein-specific MTase. A , targeting of METTL12 gene in human HAP1 WT cells by CRISPR/Cas9 generated METTL12 KO cells containing a 1 base pair insertion in the METTL12 gene, located upstream of motif Post I, resulting in generation of truncated METTL12 protein. The dashed lines interrupting the open reading frames correspond to 177 nucleotides, i.e. 59 amino acids. B , METTL12-dependent protein methylation in cell extracts. Mitochondrial extracts from HAP1 WT or METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12. Methylation reactions were separated by SDS-PAGE and transferred to a membrane. Methylation was visualized by fluorography ( top ) of the Ponceau S-stained membrane ( bottom ). Arrows indicate the positions of the ∼48 kDa substrate and METTL12. C , D107A mutation abrogates enzymatic activity of METTL12. Mitochondrial extracts from METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12, either WT or D107A-mutated. Methylation was analyzed as in B . Note: in panels B and C different levels of background (non-METTL12-dependent) methylation are observed; this is likely due to differences in the purity of the mitochondrial extracts.

    Journal: The Journal of Biological Chemistry

    Article Title: Uncovering human METTL12 as a mitochondrial methyltransferase that modulates citrate synthase activity through metabolite-sensitive lysine methylation

    doi: 10.1074/jbc.M117.808451

    Figure Lengend Snippet: Human METTL12 is a protein-specific MTase. A , targeting of METTL12 gene in human HAP1 WT cells by CRISPR/Cas9 generated METTL12 KO cells containing a 1 base pair insertion in the METTL12 gene, located upstream of motif Post I, resulting in generation of truncated METTL12 protein. The dashed lines interrupting the open reading frames correspond to 177 nucleotides, i.e. 59 amino acids. B , METTL12-dependent protein methylation in cell extracts. Mitochondrial extracts from HAP1 WT or METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12. Methylation reactions were separated by SDS-PAGE and transferred to a membrane. Methylation was visualized by fluorography ( top ) of the Ponceau S-stained membrane ( bottom ). Arrows indicate the positions of the ∼48 kDa substrate and METTL12. C , D107A mutation abrogates enzymatic activity of METTL12. Mitochondrial extracts from METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12, either WT or D107A-mutated. Methylation was analyzed as in B . Note: in panels B and C different levels of background (non-METTL12-dependent) methylation are observed; this is likely due to differences in the purity of the mitochondrial extracts.

    Article Snippet: For scintillation counting and titration experiments, MTase reactions contained [3 H]AdoMet, which was diluted with nonradioactive AdoMet (New England BioLabs).

    Techniques: CRISPR, Generated, Methylation, Incubation, Recombinant, SDS Page, Staining, Mutagenesis, Activity Assay

    Sequence analysis and structural modelling of Ynl024c. (A) Topology diagram of the canonical 7BS MTase fold. Arrows and rectangles indicate β-strands and α-helices, respectively. The seven β-strands are designated “1–7”, the α-helices connecting them “A-F”, and the secondary structure elements preceding the 7BS-fold denoted “Z”. B, Protein sequence alignment of Ynl024c and human METTL21 proteins. Motifs “I”, “Post I” and “II”, which are shared by all 7BS MTases, as well as the DXXY motif, a hallmark of MTF16, are indicated by boxes. Above the alignment are indicated the secondary structure elements from the solved crystal structures of VCP-KMT (red; pdb 4LG1), METTL21A (green; pdb 4LEC), METTL21B (blue; pdb 4QPN) and METTL21C (orange; pdb 4MTL), a predicted Ynl024c structure (black; see also (C)), as well as a secondary structure prediction for Ynl024c, performed with Jpred 3 (dashed, black). β-strands and α-helices are indicated by arrows and thick lines, respectively, and the numbering/lettering of these are as outlined in A). Asterisks indicate conserved active site residues represented in (D). (C) Predicted structural model of Ynl024c. The model was generated by one-to-one threading with Phyre2, using METTL21A as a template. (D) localization of putatively important catalytic residues (green) in the active site of the Ynl024c structural model. AdoMet is shown in purple. The shown residues are indicated by asterisk in the sequence alignment in (B). (E) Structural alignment of human VCP-KMT, METTL21A, METTL21B and METTL21C (Color code as in (B)).

    Journal: PLoS ONE

    Article Title: Saccharomyces cerevisiae Eukaryotic Elongation Factor 1A (eEF1A) Is Methylated at Lys-390 by a METTL21-Like Methyltransferase

    doi: 10.1371/journal.pone.0131426

    Figure Lengend Snippet: Sequence analysis and structural modelling of Ynl024c. (A) Topology diagram of the canonical 7BS MTase fold. Arrows and rectangles indicate β-strands and α-helices, respectively. The seven β-strands are designated “1–7”, the α-helices connecting them “A-F”, and the secondary structure elements preceding the 7BS-fold denoted “Z”. B, Protein sequence alignment of Ynl024c and human METTL21 proteins. Motifs “I”, “Post I” and “II”, which are shared by all 7BS MTases, as well as the DXXY motif, a hallmark of MTF16, are indicated by boxes. Above the alignment are indicated the secondary structure elements from the solved crystal structures of VCP-KMT (red; pdb 4LG1), METTL21A (green; pdb 4LEC), METTL21B (blue; pdb 4QPN) and METTL21C (orange; pdb 4MTL), a predicted Ynl024c structure (black; see also (C)), as well as a secondary structure prediction for Ynl024c, performed with Jpred 3 (dashed, black). β-strands and α-helices are indicated by arrows and thick lines, respectively, and the numbering/lettering of these are as outlined in A). Asterisks indicate conserved active site residues represented in (D). (C) Predicted structural model of Ynl024c. The model was generated by one-to-one threading with Phyre2, using METTL21A as a template. (D) localization of putatively important catalytic residues (green) in the active site of the Ynl024c structural model. AdoMet is shown in purple. The shown residues are indicated by asterisk in the sequence alignment in (B). (E) Structural alignment of human VCP-KMT, METTL21A, METTL21B and METTL21C (Color code as in (B)).

    Article Snippet: For MS analysis radiolabeled AdoMet was replaced by 1.2 mM unlabeled AdoMet (NEB) and MTase reactions were terminated by boiling in NuPAGE buffer (Life Technologies).

    Techniques: Sequencing, Generated

    In vitro MTase activity of Ynl024c. Protein extract from the ynl024cΔ yeast strain (denoted “ Sc ynl024c Δ”) was treated with a protein extract from E . coli either expressing 6xHis-tagged Ynl024c from the pET28a- YNL024C plasmid (denoted “ Ec + Ynl024c”) or devoid of an expression plasmid (denoted “ Ec ”), at 37°C for 60 min in the presence of [ 3 H]AdoMet, and 1 mM GTP where indicated. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane and then subjected to Ponceau S staining (bottom) and fluorograpy (top).

    Journal: PLoS ONE

    Article Title: Saccharomyces cerevisiae Eukaryotic Elongation Factor 1A (eEF1A) Is Methylated at Lys-390 by a METTL21-Like Methyltransferase

    doi: 10.1371/journal.pone.0131426

    Figure Lengend Snippet: In vitro MTase activity of Ynl024c. Protein extract from the ynl024cΔ yeast strain (denoted “ Sc ynl024c Δ”) was treated with a protein extract from E . coli either expressing 6xHis-tagged Ynl024c from the pET28a- YNL024C plasmid (denoted “ Ec + Ynl024c”) or devoid of an expression plasmid (denoted “ Ec ”), at 37°C for 60 min in the presence of [ 3 H]AdoMet, and 1 mM GTP where indicated. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane and then subjected to Ponceau S staining (bottom) and fluorograpy (top).

    Article Snippet: For MS analysis radiolabeled AdoMet was replaced by 1.2 mM unlabeled AdoMet (NEB) and MTase reactions were terminated by boiling in NuPAGE buffer (Life Technologies).

    Techniques: In Vitro, Activity Assay, Expressing, Plasmid Preparation, SDS Page, Staining

    Identification of METTL13 as an eEF1A-specific methyltransferase. a Workflow of mass spectrometry-based quantitative peptide pull-down screen. Synthetic peptides corresponding N-terminally trimethylated (Nt-Me3) and unmethylated (Nt-Me0) eEF1A were used as baits to enrich proteins from HAP-1 cell extracts. b . c Domain organization of METTL13. The boundaries for used constructs encompassing the N-terminal (MT13-N) and the C-terminal (MT13-C) methyltransferase domains are indicated. d , e Evaluation of METTL13 constructs for eEF1A-specific methyltransferase activity. MT13-N ( d ) and MT13-C ( e ) were incubated with [ 3 H]-AdoMet and eEF1A1 carrying an N-terminal or C-terminal His-tag in the absence of cofactors and in the presence of either GDP or GTP. Methylation was visualized by fluorography (top panels) and the membranes were stained with Ponceau S (bottom panels) to assess protein loading

    Journal: Nature Communications

    Article Title: The dual methyltransferase METTL13 targets N terminus and Lys55 of eEF1A and modulates codon-specific translation rates

    doi: 10.1038/s41467-018-05646-y

    Figure Lengend Snippet: Identification of METTL13 as an eEF1A-specific methyltransferase. a Workflow of mass spectrometry-based quantitative peptide pull-down screen. Synthetic peptides corresponding N-terminally trimethylated (Nt-Me3) and unmethylated (Nt-Me0) eEF1A were used as baits to enrich proteins from HAP-1 cell extracts. b . c Domain organization of METTL13. The boundaries for used constructs encompassing the N-terminal (MT13-N) and the C-terminal (MT13-C) methyltransferase domains are indicated. d , e Evaluation of METTL13 constructs for eEF1A-specific methyltransferase activity. MT13-N ( d ) and MT13-C ( e ) were incubated with [ 3 H]-AdoMet and eEF1A1 carrying an N-terminal or C-terminal His-tag in the absence of cofactors and in the presence of either GDP or GTP. Methylation was visualized by fluorography (top panels) and the membranes were stained with Ponceau S (bottom panels) to assess protein loading

    Article Snippet: For quantitative MTase assays, [3 H]-AdoMet was diluted with non-radioactive AdoMet (New England Biolabs) ([AdoMet]total = 32.6 μM) .

    Techniques: Mass Spectrometry, Construct, Activity Assay, Incubation, Methylation, Staining