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    New England Biolabs nonradioactive s adenosylmethionine
    G9a and GLP are methylated on their N‐terminal domain in cells Schematic representation of the related proteins GLP (EHMT1) and G9a (EHMT2). N: N‐terminal coactivator domain, E: polyglutamate domain, Cys: cysteine‐rich region, ANK: six ankyrin repeats, SET: SET‐domain containing methyltransferase activity. Partial protein sequence of hG9a and hGLP homologs shows the hypothetical methylated lysine residues (K) in red. After protein methylation reactions, in vitro methylated proteins were detected by immunoblot with pan‐methyllysine antibody (pan met‐K). The corresponding Coomassie‐stained gels are shown as loading controls. <t>SAM,</t> <t>S‐adenosylmethionine.</t> Cos‐7 cells were transfected with plasmids encoding full‐length HA‐hG9a wild type or K185R mutant, or full‐length HA‐hGLP wild type or K205R mutant. Lysates were immunoprecipitated (IP) with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Expression of HA‐tagged proteins and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input). Cos‐7 cells were transfected with a plasmid encoding full‐length HA‐hG9a and treated with 2 μM UNC0646 or vehicle DMSO for 24 h. Lysates were immunoprecipitated with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Methylation and phosphorylation of endogenous G9a and GLP in A549 cells treated with 100 nM dex for 4 h were analyzed by immunoprecipitation with control IgG antibody, anti‐G9a (top), or anti‐GLP (bottom), followed by immunoblot with antibodies listed. Expression of G9a, GLP, and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input).
    Nonradioactive S Adenosylmethionine, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    New England Biolabs adomet
    Mg18 2′O MTase activity. <t>AdoMet-dependent</t> MTase assays were performed on equimolar amounts of short capped RNAs substrates (GpppAN 13 ), N7- or 2′O-methylated capped <t>RNA</t> substrates ( 7Me GpppAN 13 or GpppA 2′OMe N 13 ), A. castellanii mRNAs or homopolymeric poly (U), (C), (G) and (A). Human N7 MTase and Vaccinia virus VP39 were use as controls.
    Adomet, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    PerkinElmer adomet
    Stick model of RNA bound to the nsp16 RNA binding groove and <t>Sinefungin</t> in the methyltransferase active site. A) In this representation, Sinefungin was preferred over SAH because one of its NH 2 groups approximates the direction of a transferred CH 3 group from the SAM substrate. Carbon is white, oxygen is red, nitrogen is blue and phosphorous is orange. Nsp16 and nsp10 are rendered as a solvent-accessible surface colored grey and wheat respectively. The Sinefungin molecule defines the methyltransferase active site. Missing residues in the 135-137 loop (see text) are indicated by a shaded blue dotted box. Position of Y30 and Y132 are indicated. Y30 generated poor electron density (see « Methods ») and its aromatic ring position has been manually adjusted before generation of this image. B) Close caption of the methyltransferase active site showing distance between the NH2 of Sinefungin to the 2′-O of the ribose of the first base, thus mimicking the position of the methyl of the <t>S-adenosylmethionine.</t>
    Adomet, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 92/100, based on 225 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    G9a and GLP are methylated on their N‐terminal domain in cells Schematic representation of the related proteins GLP (EHMT1) and G9a (EHMT2). N: N‐terminal coactivator domain, E: polyglutamate domain, Cys: cysteine‐rich region, ANK: six ankyrin repeats, SET: SET‐domain containing methyltransferase activity. Partial protein sequence of hG9a and hGLP homologs shows the hypothetical methylated lysine residues (K) in red. After protein methylation reactions, in vitro methylated proteins were detected by immunoblot with pan‐methyllysine antibody (pan met‐K). The corresponding Coomassie‐stained gels are shown as loading controls. SAM, S‐adenosylmethionine. Cos‐7 cells were transfected with plasmids encoding full‐length HA‐hG9a wild type or K185R mutant, or full‐length HA‐hGLP wild type or K205R mutant. Lysates were immunoprecipitated (IP) with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Expression of HA‐tagged proteins and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input). Cos‐7 cells were transfected with a plasmid encoding full‐length HA‐hG9a and treated with 2 μM UNC0646 or vehicle DMSO for 24 h. Lysates were immunoprecipitated with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Methylation and phosphorylation of endogenous G9a and GLP in A549 cells treated with 100 nM dex for 4 h were analyzed by immunoprecipitation with control IgG antibody, anti‐G9a (top), or anti‐GLP (bottom), followed by immunoblot with antibodies listed. Expression of G9a, GLP, and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input).

    Journal: EMBO Reports

    Article Title: A post‐translational modification switch controls coactivator function of histone methyltransferases G9a and GLP

    doi: 10.15252/embr.201744060

    Figure Lengend Snippet: G9a and GLP are methylated on their N‐terminal domain in cells Schematic representation of the related proteins GLP (EHMT1) and G9a (EHMT2). N: N‐terminal coactivator domain, E: polyglutamate domain, Cys: cysteine‐rich region, ANK: six ankyrin repeats, SET: SET‐domain containing methyltransferase activity. Partial protein sequence of hG9a and hGLP homologs shows the hypothetical methylated lysine residues (K) in red. After protein methylation reactions, in vitro methylated proteins were detected by immunoblot with pan‐methyllysine antibody (pan met‐K). The corresponding Coomassie‐stained gels are shown as loading controls. SAM, S‐adenosylmethionine. Cos‐7 cells were transfected with plasmids encoding full‐length HA‐hG9a wild type or K185R mutant, or full‐length HA‐hGLP wild type or K205R mutant. Lysates were immunoprecipitated (IP) with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Expression of HA‐tagged proteins and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input). Cos‐7 cells were transfected with a plasmid encoding full‐length HA‐hG9a and treated with 2 μM UNC0646 or vehicle DMSO for 24 h. Lysates were immunoprecipitated with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Methylation and phosphorylation of endogenous G9a and GLP in A549 cells treated with 100 nM dex for 4 h were analyzed by immunoprecipitation with control IgG antibody, anti‐G9a (top), or anti‐GLP (bottom), followed by immunoblot with antibodies listed. Expression of G9a, GLP, and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input).

    Article Snippet: Bacterially produced GST fusion proteins (2 μg) of N‐terminal fragments of G9a or GLP (GST‐hG9a N or GST‐hGLP N), mutant (GST‐hG9a N K185R or GST‐hGLP N K205R or GST‐hG9a N T186A), or GST alone were incubated 90 min at 30°C with GST‐hG9a ΔN or GST‐hGLP ΔN in the presence (or not) of 1 mM of unradiolabeled SAM (New England Biolabs, B9003S).

    Techniques: Methylation, Activity Assay, Sequencing, In Vitro, Staining, Transfection, Mutagenesis, Immunoprecipitation, Expressing, Plasmid Preparation

    Mg18 2′O MTase activity. AdoMet-dependent MTase assays were performed on equimolar amounts of short capped RNAs substrates (GpppAN 13 ), N7- or 2′O-methylated capped RNA substrates ( 7Me GpppAN 13 or GpppA 2′OMe N 13 ), A. castellanii mRNAs or homopolymeric poly (U), (C), (G) and (A). Human N7 MTase and Vaccinia virus VP39 were use as controls.

    Journal: Nucleic Acids Research

    Article Title: mRNA maturation in giant viruses: variation on a theme

    doi: 10.1093/nar/gkv224

    Figure Lengend Snippet: Mg18 2′O MTase activity. AdoMet-dependent MTase assays were performed on equimolar amounts of short capped RNAs substrates (GpppAN 13 ), N7- or 2′O-methylated capped RNA substrates ( 7Me GpppAN 13 or GpppA 2′OMe N 13 ), A. castellanii mRNAs or homopolymeric poly (U), (C), (G) and (A). Human N7 MTase and Vaccinia virus VP39 were use as controls.

    Article Snippet: MTase activity assays were performed at 30°C in 40 mM Tris-HCl pH 7.5, 1 mM DTT, 1 mM MgCl2 , 0.7 μM RNA substrates, 10 μM AdoMet (NEB), and 0.03 mCi/ml 3 H-AdoMet (GE Healthcare).

    Techniques: Activity Assay, Methylation

    Stick model of RNA bound to the nsp16 RNA binding groove and Sinefungin in the methyltransferase active site. A) In this representation, Sinefungin was preferred over SAH because one of its NH 2 groups approximates the direction of a transferred CH 3 group from the SAM substrate. Carbon is white, oxygen is red, nitrogen is blue and phosphorous is orange. Nsp16 and nsp10 are rendered as a solvent-accessible surface colored grey and wheat respectively. The Sinefungin molecule defines the methyltransferase active site. Missing residues in the 135-137 loop (see text) are indicated by a shaded blue dotted box. Position of Y30 and Y132 are indicated. Y30 generated poor electron density (see « Methods ») and its aromatic ring position has been manually adjusted before generation of this image. B) Close caption of the methyltransferase active site showing distance between the NH2 of Sinefungin to the 2′-O of the ribose of the first base, thus mimicking the position of the methyl of the S-adenosylmethionine.

    Journal: PLoS Pathogens

    Article Title: Crystal Structure and Functional Analysis of the SARS-Coronavirus RNA Cap 2?-O-Methyltransferase nsp10/nsp16 Complex

    doi: 10.1371/journal.ppat.1002059

    Figure Lengend Snippet: Stick model of RNA bound to the nsp16 RNA binding groove and Sinefungin in the methyltransferase active site. A) In this representation, Sinefungin was preferred over SAH because one of its NH 2 groups approximates the direction of a transferred CH 3 group from the SAM substrate. Carbon is white, oxygen is red, nitrogen is blue and phosphorous is orange. Nsp16 and nsp10 are rendered as a solvent-accessible surface colored grey and wheat respectively. The Sinefungin molecule defines the methyltransferase active site. Missing residues in the 135-137 loop (see text) are indicated by a shaded blue dotted box. Position of Y30 and Y132 are indicated. Y30 generated poor electron density (see « Methods ») and its aromatic ring position has been manually adjusted before generation of this image. B) Close caption of the methyltransferase active site showing distance between the NH2 of Sinefungin to the 2′-O of the ribose of the first base, thus mimicking the position of the methyl of the S-adenosylmethionine.

    Article Snippet: Reagents AdoMet and cap analogs GpppA and 7Me GpppA were purchased from New England BioLabs, the[3 H]-AdoMet was purchased from Perkin Elmer and Sinefungin (adenosylornithine) from Sigma-Aldrich.

    Techniques: RNA Binding Assay, Generated