γ 32 p atp New England Biolabs Search Results


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  • 99
    Qiagen qiaquick nucleotide removal kit
    Qiaquick Nucleotide Removal Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 3529 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 3529 article reviews
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    99
    New England Biolabs terminal transferase
    Terminal Transferase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 816 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    New England Biolabs γ32 p atp
    γ32 P Atp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 495 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 495 article reviews
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    99
    New England Biolabs polynucleotide t4 kinase
    Polynucleotide T4 Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 63 article reviews
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    97
    PerkinElmer γ32 p atp
    γ32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 97/100, based on 1107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    PerkinElmer α32 p datp
    α32 P Datp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 polynucleotide kinase
    Identification of nucleotidyl adduct. ( A ) MALDI-TOF mass spectrometry of input substrate d3.1 (top left), <t>5΄-thiophosphorylated</t> d3.1 that was generated by treatment with ATPγS and <t>PNK</t> (bottom left), ribozyme-catalyzed product that was formed by treatment of d3.1 with K28min and GTPγS (top right), and acid-decomposition of the ribozyme-catalyzed product (bottom right). The M–H peaks produced by each treatment are indicated: blue triangles, 6784.28 m/z (expected 6782.17 a.m.u for input d3.1 substrate); magenta triangles, 6880.38 m/z (expected 6878.11 a.m.u for mono-thiophosphorylated d3.1); green triangles, 7322.00 m/z (expected 7319.10 a.m.u for adduct shown in panel F top); red triangles, 7225.3 m/z (expected 7223.16 a.m.u for adduct shown in panel F bottom). ( B ) Incorporation of α- 32 P label into unlabeled acceptor strand. Controls in first two lanes show 5΄- 32 P-labeled d3.1 substrate alone (NR) or incubated with ribozyme K28min and GTPγS (sP). For remaining lanes, non-radiolabeled d3.1 substrate was incubated with ribozyme K28min and [α- 32 P]GTP for 5 min before adding the indicated amount of unlabeled GTP. Reactions were performed at 10°C for 18 h. ( C ) Thiophosphorylation reactions containing the indicated combinations of ribozyme, substrate and donor were treated with sodium periodate, followed by amination with Cy3 hydrazide dye. For the sample in Lane 4, the product of the ribozyme-catalyzed reaction was purified from an APM gel prior to the dye labeling reaction. Gel was scanned for Cy3 fluorescence (Ex. 532 nm Em. 570 nm). Phosphorimages are of 20% denaturing PAGE.
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 24303 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    t4 polynucleotide kinase - by Bioz Stars, 2020-04
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    91
    GE Healthcare γ32 p atp
    Identification of nucleotidyl adduct. ( A ) MALDI-TOF mass spectrometry of input substrate d3.1 (top left), <t>5΄-thiophosphorylated</t> d3.1 that was generated by treatment with ATPγS and <t>PNK</t> (bottom left), ribozyme-catalyzed product that was formed by treatment of d3.1 with K28min and GTPγS (top right), and acid-decomposition of the ribozyme-catalyzed product (bottom right). The M–H peaks produced by each treatment are indicated: blue triangles, 6784.28 m/z (expected 6782.17 a.m.u for input d3.1 substrate); magenta triangles, 6880.38 m/z (expected 6878.11 a.m.u for mono-thiophosphorylated d3.1); green triangles, 7322.00 m/z (expected 7319.10 a.m.u for adduct shown in panel F top); red triangles, 7225.3 m/z (expected 7223.16 a.m.u for adduct shown in panel F bottom). ( B ) Incorporation of α- 32 P label into unlabeled acceptor strand. Controls in first two lanes show 5΄- 32 P-labeled d3.1 substrate alone (NR) or incubated with ribozyme K28min and GTPγS (sP). For remaining lanes, non-radiolabeled d3.1 substrate was incubated with ribozyme K28min and [α- 32 P]GTP for 5 min before adding the indicated amount of unlabeled GTP. Reactions were performed at 10°C for 18 h. ( C ) Thiophosphorylation reactions containing the indicated combinations of ribozyme, substrate and donor were treated with sodium periodate, followed by amination with Cy3 hydrazide dye. For the sample in Lane 4, the product of the ribozyme-catalyzed reaction was purified from an APM gel prior to the dye labeling reaction. Gel was scanned for Cy3 fluorescence (Ex. 532 nm Em. 570 nm). Phosphorimages are of 20% denaturing PAGE.
    γ32 P Atp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 756 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    γ32 p atp - by Bioz Stars, 2020-04
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    92
    PerkinElmer α32 p atp
    Identification of nucleotidyl adduct. ( A ) MALDI-TOF mass spectrometry of input substrate d3.1 (top left), <t>5΄-thiophosphorylated</t> d3.1 that was generated by treatment with ATPγS and <t>PNK</t> (bottom left), ribozyme-catalyzed product that was formed by treatment of d3.1 with K28min and GTPγS (top right), and acid-decomposition of the ribozyme-catalyzed product (bottom right). The M–H peaks produced by each treatment are indicated: blue triangles, 6784.28 m/z (expected 6782.17 a.m.u for input d3.1 substrate); magenta triangles, 6880.38 m/z (expected 6878.11 a.m.u for mono-thiophosphorylated d3.1); green triangles, 7322.00 m/z (expected 7319.10 a.m.u for adduct shown in panel F top); red triangles, 7225.3 m/z (expected 7223.16 a.m.u for adduct shown in panel F bottom). ( B ) Incorporation of α- 32 P label into unlabeled acceptor strand. Controls in first two lanes show 5΄- 32 P-labeled d3.1 substrate alone (NR) or incubated with ribozyme K28min and GTPγS (sP). For remaining lanes, non-radiolabeled d3.1 substrate was incubated with ribozyme K28min and [α- 32 P]GTP for 5 min before adding the indicated amount of unlabeled GTP. Reactions were performed at 10°C for 18 h. ( C ) Thiophosphorylation reactions containing the indicated combinations of ribozyme, substrate and donor were treated with sodium periodate, followed by amination with Cy3 hydrazide dye. For the sample in Lane 4, the product of the ribozyme-catalyzed reaction was purified from an APM gel prior to the dye labeling reaction. Gel was scanned for Cy3 fluorescence (Ex. 532 nm Em. 570 nm). Phosphorimages are of 20% denaturing PAGE.
    α32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 92/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    α32 p atp - by Bioz Stars, 2020-04
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    99
    New England Biolabs t4 dna ligase
    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and <t>T4</t> DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 38631 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    HARTMANN ANALYTIC γ32 p atp
    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and <t>T4</t> DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.
    γ32 P Atp, supplied by HARTMANN ANALYTIC, used in various techniques. Bioz Stars score: 94/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Valiant γ32 p atp
    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and <t>T4</t> DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.
    γ32 P Atp, supplied by Valiant, used in various techniques. Bioz Stars score: 87/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare dntps
    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and <t>T4</t> DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.
    Dntps, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 2063 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs camp dependent protein kinase
    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and <t>T4</t> DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.
    Camp Dependent Protein Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs antarctic phosphatase
    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and <t>T4</t> DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.
    Antarctic Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4986 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 4986 article reviews
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    antarctic phosphatase - by Bioz Stars, 2020-04
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    90
    GE Healthcare α32 p ntps
    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and <t>T4</t> DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.
    α32 P Ntps, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs oligos
    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and <t>T4</t> DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.
    Oligos, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    oligos - by Bioz Stars, 2020-04
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    93
    PerkinElmer ci mmole
    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and <t>T4</t> DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.
    Ci Mmole, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 93/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ci mmole - by Bioz Stars, 2020-04
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    94
    New England Biolabs t4 kinase
    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and <t>T4</t> DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.
    T4 Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 520 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs reaction buffer
    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and <t>T4</t> DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.
    Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1347 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    reaction buffer - by Bioz Stars, 2020-04
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    90
    New England Biolabs kinase buffer
    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and <t>T4</t> DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.
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    99
    New England Biolabs calf intestinal alkaline phosphatase
    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and <t>T4</t> DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.
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    99
    New England Biolabs ecori
    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and <t>T4</t> DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.
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    95
    Bio-Rad microbiospin 6 columns
    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and <t>T4</t> DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.
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    92
    New England Biolabs ck2 enzyme
    Inositol pyrophosphates pyrophosphorylate dynein IC. ( A ) Bacterially expressed and purified IC(1–111) was phosphorylated in vitro by <t>CK2,</t> and phosphosite identification was contracted out to the Taplin Mass Spectrometry Facility, Harvard Medical School. The MS/MS spectrum is shown for the doubly phosphorylated peptide corresponding to residues 42–55 of mouse IC-2C (EAAVpSVQEEpSDLEK). The sequence shows the peptide fragmentation pattern, and the table shows masses of all b and y ions, highlighting the ions obtained in the spectrum. Arrows indicate fragment ions containing phosphorylated Ser residues. The mass of fragment y5 indicates phosphorylation of Ser51, and the masses of y10 and b10 correspond to phosphorylation of Ser46 and Ser51. ( B ) Bacterially expressed and purified GST or GST-tagged IC(1–70), IC(1–111), and IC(1–111)S51A were prephosphorylated with CK2 and unlabeled ATP and incubated with 5[β- 32 P]IP 7 . Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and the proteins were detected by Ponceau S staining (left). The phosphorimager scan was subjected to ‘levels’ adjustment in Adobe Photoshop to improve visualization. The image intensity of the pyrophosphorylated protein was normalized to the corresponding total protein. The pyrophosphorylation intensity of each IC fragment was compared with GST. Data are mean ± range from two independent experiments. ( C ) Back-pyrophosphorylation of endogenous dynein IC by IP 7 . Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with 5[β- 32 P]IP 7 , resolved by NuPAGE, and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right), and proteins were detected by western blotting (left). The image intensity of the pyrophosphorylated protein was normalized to the corresponding immunoprecipitated protein. The fold change in the extent of pyrophosphorylation of IC in Ip6k1 −/− compared with Ip6k1 +/+ MEFs is indicated. Data are mean ± SEM from three independent experiments. ( D ) Back-phosphorylation of endogenous dynein IC by CK2. Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with CK2 and [γ- 32 P]ATP. Proteins were resolved and detected as in ( C ). The fold change in phosphorylation was calculated as in ( C ). Data are mean ± range from two independent experiments.
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    94
    New England Biolabs protein kinase buffer
    Inositol pyrophosphates pyrophosphorylate dynein IC. ( A ) Bacterially expressed and purified IC(1–111) was phosphorylated in vitro by <t>CK2,</t> and phosphosite identification was contracted out to the Taplin Mass Spectrometry Facility, Harvard Medical School. The MS/MS spectrum is shown for the doubly phosphorylated peptide corresponding to residues 42–55 of mouse IC-2C (EAAVpSVQEEpSDLEK). The sequence shows the peptide fragmentation pattern, and the table shows masses of all b and y ions, highlighting the ions obtained in the spectrum. Arrows indicate fragment ions containing phosphorylated Ser residues. The mass of fragment y5 indicates phosphorylation of Ser51, and the masses of y10 and b10 correspond to phosphorylation of Ser46 and Ser51. ( B ) Bacterially expressed and purified GST or GST-tagged IC(1–70), IC(1–111), and IC(1–111)S51A were prephosphorylated with CK2 and unlabeled ATP and incubated with 5[β- 32 P]IP 7 . Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and the proteins were detected by Ponceau S staining (left). The phosphorimager scan was subjected to ‘levels’ adjustment in Adobe Photoshop to improve visualization. The image intensity of the pyrophosphorylated protein was normalized to the corresponding total protein. The pyrophosphorylation intensity of each IC fragment was compared with GST. Data are mean ± range from two independent experiments. ( C ) Back-pyrophosphorylation of endogenous dynein IC by IP 7 . Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with 5[β- 32 P]IP 7 , resolved by NuPAGE, and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right), and proteins were detected by western blotting (left). The image intensity of the pyrophosphorylated protein was normalized to the corresponding immunoprecipitated protein. The fold change in the extent of pyrophosphorylation of IC in Ip6k1 −/− compared with Ip6k1 +/+ MEFs is indicated. Data are mean ± SEM from three independent experiments. ( D ) Back-phosphorylation of endogenous dynein IC by CK2. Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with CK2 and [γ- 32 P]ATP. Proteins were resolved and detected as in ( C ). The fold change in phosphorylation was calculated as in ( C ). Data are mean ± range from two independent experiments.
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    91
    GE Healthcare g 50 spin columns
    Inositol pyrophosphates pyrophosphorylate dynein IC. ( A ) Bacterially expressed and purified IC(1–111) was phosphorylated in vitro by <t>CK2,</t> and phosphosite identification was contracted out to the Taplin Mass Spectrometry Facility, Harvard Medical School. The MS/MS spectrum is shown for the doubly phosphorylated peptide corresponding to residues 42–55 of mouse IC-2C (EAAVpSVQEEpSDLEK). The sequence shows the peptide fragmentation pattern, and the table shows masses of all b and y ions, highlighting the ions obtained in the spectrum. Arrows indicate fragment ions containing phosphorylated Ser residues. The mass of fragment y5 indicates phosphorylation of Ser51, and the masses of y10 and b10 correspond to phosphorylation of Ser46 and Ser51. ( B ) Bacterially expressed and purified GST or GST-tagged IC(1–70), IC(1–111), and IC(1–111)S51A were prephosphorylated with CK2 and unlabeled ATP and incubated with 5[β- 32 P]IP 7 . Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and the proteins were detected by Ponceau S staining (left). The phosphorimager scan was subjected to ‘levels’ adjustment in Adobe Photoshop to improve visualization. The image intensity of the pyrophosphorylated protein was normalized to the corresponding total protein. The pyrophosphorylation intensity of each IC fragment was compared with GST. Data are mean ± range from two independent experiments. ( C ) Back-pyrophosphorylation of endogenous dynein IC by IP 7 . Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with 5[β- 32 P]IP 7 , resolved by NuPAGE, and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right), and proteins were detected by western blotting (left). The image intensity of the pyrophosphorylated protein was normalized to the corresponding immunoprecipitated protein. The fold change in the extent of pyrophosphorylation of IC in Ip6k1 −/− compared with Ip6k1 +/+ MEFs is indicated. Data are mean ± SEM from three independent experiments. ( D ) Back-phosphorylation of endogenous dynein IC by CK2. Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with CK2 and [γ- 32 P]ATP. Proteins were resolved and detected as in ( C ). The fold change in phosphorylation was calculated as in ( C ). Data are mean ± range from two independent experiments.
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    87
    GE Healthcare g25 probe quant columns
    Inositol pyrophosphates pyrophosphorylate dynein IC. ( A ) Bacterially expressed and purified IC(1–111) was phosphorylated in vitro by <t>CK2,</t> and phosphosite identification was contracted out to the Taplin Mass Spectrometry Facility, Harvard Medical School. The MS/MS spectrum is shown for the doubly phosphorylated peptide corresponding to residues 42–55 of mouse IC-2C (EAAVpSVQEEpSDLEK). The sequence shows the peptide fragmentation pattern, and the table shows masses of all b and y ions, highlighting the ions obtained in the spectrum. Arrows indicate fragment ions containing phosphorylated Ser residues. The mass of fragment y5 indicates phosphorylation of Ser51, and the masses of y10 and b10 correspond to phosphorylation of Ser46 and Ser51. ( B ) Bacterially expressed and purified GST or GST-tagged IC(1–70), IC(1–111), and IC(1–111)S51A were prephosphorylated with CK2 and unlabeled ATP and incubated with 5[β- 32 P]IP 7 . Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and the proteins were detected by Ponceau S staining (left). The phosphorimager scan was subjected to ‘levels’ adjustment in Adobe Photoshop to improve visualization. The image intensity of the pyrophosphorylated protein was normalized to the corresponding total protein. The pyrophosphorylation intensity of each IC fragment was compared with GST. Data are mean ± range from two independent experiments. ( C ) Back-pyrophosphorylation of endogenous dynein IC by IP 7 . Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with 5[β- 32 P]IP 7 , resolved by NuPAGE, and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right), and proteins were detected by western blotting (left). The image intensity of the pyrophosphorylated protein was normalized to the corresponding immunoprecipitated protein. The fold change in the extent of pyrophosphorylation of IC in Ip6k1 −/− compared with Ip6k1 +/+ MEFs is indicated. Data are mean ± SEM from three independent experiments. ( D ) Back-phosphorylation of endogenous dynein IC by CK2. Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with CK2 and [γ- 32 P]ATP. Proteins were resolved and detected as in ( C ). The fold change in phosphorylation was calculated as in ( C ). Data are mean ± range from two independent experiments.
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    99
    Thermo Fisher atp
    Inositol pyrophosphates pyrophosphorylate dynein IC. ( A ) Bacterially expressed and purified IC(1–111) was phosphorylated in vitro by <t>CK2,</t> and phosphosite identification was contracted out to the Taplin Mass Spectrometry Facility, Harvard Medical School. The MS/MS spectrum is shown for the doubly phosphorylated peptide corresponding to residues 42–55 of mouse IC-2C (EAAVpSVQEEpSDLEK). The sequence shows the peptide fragmentation pattern, and the table shows masses of all b and y ions, highlighting the ions obtained in the spectrum. Arrows indicate fragment ions containing phosphorylated Ser residues. The mass of fragment y5 indicates phosphorylation of Ser51, and the masses of y10 and b10 correspond to phosphorylation of Ser46 and Ser51. ( B ) Bacterially expressed and purified GST or GST-tagged IC(1–70), IC(1–111), and IC(1–111)S51A were prephosphorylated with CK2 and unlabeled ATP and incubated with 5[β- 32 P]IP 7 . Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and the proteins were detected by Ponceau S staining (left). The phosphorimager scan was subjected to ‘levels’ adjustment in Adobe Photoshop to improve visualization. The image intensity of the pyrophosphorylated protein was normalized to the corresponding total protein. The pyrophosphorylation intensity of each IC fragment was compared with GST. Data are mean ± range from two independent experiments. ( C ) Back-pyrophosphorylation of endogenous dynein IC by IP 7 . Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with 5[β- 32 P]IP 7 , resolved by NuPAGE, and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right), and proteins were detected by western blotting (left). The image intensity of the pyrophosphorylated protein was normalized to the corresponding immunoprecipitated protein. The fold change in the extent of pyrophosphorylation of IC in Ip6k1 −/− compared with Ip6k1 +/+ MEFs is indicated. Data are mean ± SEM from three independent experiments. ( D ) Back-phosphorylation of endogenous dynein IC by CK2. Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with CK2 and [γ- 32 P]ATP. Proteins were resolved and detected as in ( C ). The fold change in phosphorylation was calculated as in ( C ). Data are mean ± range from two independent experiments.
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    Zymo Research oligo clean concentrator kit
    Inositol pyrophosphates pyrophosphorylate dynein IC. ( A ) Bacterially expressed and purified IC(1–111) was phosphorylated in vitro by <t>CK2,</t> and phosphosite identification was contracted out to the Taplin Mass Spectrometry Facility, Harvard Medical School. The MS/MS spectrum is shown for the doubly phosphorylated peptide corresponding to residues 42–55 of mouse IC-2C (EAAVpSVQEEpSDLEK). The sequence shows the peptide fragmentation pattern, and the table shows masses of all b and y ions, highlighting the ions obtained in the spectrum. Arrows indicate fragment ions containing phosphorylated Ser residues. The mass of fragment y5 indicates phosphorylation of Ser51, and the masses of y10 and b10 correspond to phosphorylation of Ser46 and Ser51. ( B ) Bacterially expressed and purified GST or GST-tagged IC(1–70), IC(1–111), and IC(1–111)S51A were prephosphorylated with CK2 and unlabeled ATP and incubated with 5[β- 32 P]IP 7 . Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and the proteins were detected by Ponceau S staining (left). The phosphorimager scan was subjected to ‘levels’ adjustment in Adobe Photoshop to improve visualization. The image intensity of the pyrophosphorylated protein was normalized to the corresponding total protein. The pyrophosphorylation intensity of each IC fragment was compared with GST. Data are mean ± range from two independent experiments. ( C ) Back-pyrophosphorylation of endogenous dynein IC by IP 7 . Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with 5[β- 32 P]IP 7 , resolved by NuPAGE, and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right), and proteins were detected by western blotting (left). The image intensity of the pyrophosphorylated protein was normalized to the corresponding immunoprecipitated protein. The fold change in the extent of pyrophosphorylation of IC in Ip6k1 −/− compared with Ip6k1 +/+ MEFs is indicated. Data are mean ± SEM from three independent experiments. ( D ) Back-phosphorylation of endogenous dynein IC by CK2. Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with CK2 and [γ- 32 P]ATP. Proteins were resolved and detected as in ( C ). The fold change in phosphorylation was calculated as in ( C ). Data are mean ± range from two independent experiments.
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    Image Search Results


    Identification of nucleotidyl adduct. ( A ) MALDI-TOF mass spectrometry of input substrate d3.1 (top left), 5΄-thiophosphorylated d3.1 that was generated by treatment with ATPγS and PNK (bottom left), ribozyme-catalyzed product that was formed by treatment of d3.1 with K28min and GTPγS (top right), and acid-decomposition of the ribozyme-catalyzed product (bottom right). The M–H peaks produced by each treatment are indicated: blue triangles, 6784.28 m/z (expected 6782.17 a.m.u for input d3.1 substrate); magenta triangles, 6880.38 m/z (expected 6878.11 a.m.u for mono-thiophosphorylated d3.1); green triangles, 7322.00 m/z (expected 7319.10 a.m.u for adduct shown in panel F top); red triangles, 7225.3 m/z (expected 7223.16 a.m.u for adduct shown in panel F bottom). ( B ) Incorporation of α- 32 P label into unlabeled acceptor strand. Controls in first two lanes show 5΄- 32 P-labeled d3.1 substrate alone (NR) or incubated with ribozyme K28min and GTPγS (sP). For remaining lanes, non-radiolabeled d3.1 substrate was incubated with ribozyme K28min and [α- 32 P]GTP for 5 min before adding the indicated amount of unlabeled GTP. Reactions were performed at 10°C for 18 h. ( C ) Thiophosphorylation reactions containing the indicated combinations of ribozyme, substrate and donor were treated with sodium periodate, followed by amination with Cy3 hydrazide dye. For the sample in Lane 4, the product of the ribozyme-catalyzed reaction was purified from an APM gel prior to the dye labeling reaction. Gel was scanned for Cy3 fluorescence (Ex. 532 nm Em. 570 nm). Phosphorimages are of 20% denaturing PAGE.

    Journal: Nucleic Acids Research

    Article Title: Nucleobase modification by an RNA enzyme

    doi: 10.1093/nar/gkw1199

    Figure Lengend Snippet: Identification of nucleotidyl adduct. ( A ) MALDI-TOF mass spectrometry of input substrate d3.1 (top left), 5΄-thiophosphorylated d3.1 that was generated by treatment with ATPγS and PNK (bottom left), ribozyme-catalyzed product that was formed by treatment of d3.1 with K28min and GTPγS (top right), and acid-decomposition of the ribozyme-catalyzed product (bottom right). The M–H peaks produced by each treatment are indicated: blue triangles, 6784.28 m/z (expected 6782.17 a.m.u for input d3.1 substrate); magenta triangles, 6880.38 m/z (expected 6878.11 a.m.u for mono-thiophosphorylated d3.1); green triangles, 7322.00 m/z (expected 7319.10 a.m.u for adduct shown in panel F top); red triangles, 7225.3 m/z (expected 7223.16 a.m.u for adduct shown in panel F bottom). ( B ) Incorporation of α- 32 P label into unlabeled acceptor strand. Controls in first two lanes show 5΄- 32 P-labeled d3.1 substrate alone (NR) or incubated with ribozyme K28min and GTPγS (sP). For remaining lanes, non-radiolabeled d3.1 substrate was incubated with ribozyme K28min and [α- 32 P]GTP for 5 min before adding the indicated amount of unlabeled GTP. Reactions were performed at 10°C for 18 h. ( C ) Thiophosphorylation reactions containing the indicated combinations of ribozyme, substrate and donor were treated with sodium periodate, followed by amination with Cy3 hydrazide dye. For the sample in Lane 4, the product of the ribozyme-catalyzed reaction was purified from an APM gel prior to the dye labeling reaction. Gel was scanned for Cy3 fluorescence (Ex. 532 nm Em. 570 nm). Phosphorimages are of 20% denaturing PAGE.

    Article Snippet: Ribozymes were either transcribed using 33nM [α32 P]-CTP or non-radioactive CTP followed by 5΄ end labeled using [γ32 P]-ATP and PNK (NEB).

    Techniques: Mass Spectrometry, Generated, Produced, Labeling, Incubation, Purification, Fluorescence, Polyacrylamide Gel Electrophoresis

    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and T4 DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.

    Journal: Nucleic Acids Research

    Article Title: The Leu22Pro tumor-associated variant of DNA polymerase beta is dRP lyase deficient

    doi: 10.1093/nar/gkm1053

    Figure Lengend Snippet: L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and T4 DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.

    Article Snippet: Uracil DNA [Glycosylase (UDG) (M0280S), human AP endonuclease I (APE1) (M0282S), terminal transferase (M0252S), T4 PNK (M0201S)] and T4 DNA ligase (M0202S) were purchased from New England Biolabs.

    Techniques: Purification, Incubation, Activity Assay, Primer Extension Assay

    Inositol pyrophosphates pyrophosphorylate dynein IC. ( A ) Bacterially expressed and purified IC(1–111) was phosphorylated in vitro by CK2, and phosphosite identification was contracted out to the Taplin Mass Spectrometry Facility, Harvard Medical School. The MS/MS spectrum is shown for the doubly phosphorylated peptide corresponding to residues 42–55 of mouse IC-2C (EAAVpSVQEEpSDLEK). The sequence shows the peptide fragmentation pattern, and the table shows masses of all b and y ions, highlighting the ions obtained in the spectrum. Arrows indicate fragment ions containing phosphorylated Ser residues. The mass of fragment y5 indicates phosphorylation of Ser51, and the masses of y10 and b10 correspond to phosphorylation of Ser46 and Ser51. ( B ) Bacterially expressed and purified GST or GST-tagged IC(1–70), IC(1–111), and IC(1–111)S51A were prephosphorylated with CK2 and unlabeled ATP and incubated with 5[β- 32 P]IP 7 . Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and the proteins were detected by Ponceau S staining (left). The phosphorimager scan was subjected to ‘levels’ adjustment in Adobe Photoshop to improve visualization. The image intensity of the pyrophosphorylated protein was normalized to the corresponding total protein. The pyrophosphorylation intensity of each IC fragment was compared with GST. Data are mean ± range from two independent experiments. ( C ) Back-pyrophosphorylation of endogenous dynein IC by IP 7 . Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with 5[β- 32 P]IP 7 , resolved by NuPAGE, and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right), and proteins were detected by western blotting (left). The image intensity of the pyrophosphorylated protein was normalized to the corresponding immunoprecipitated protein. The fold change in the extent of pyrophosphorylation of IC in Ip6k1 −/− compared with Ip6k1 +/+ MEFs is indicated. Data are mean ± SEM from three independent experiments. ( D ) Back-phosphorylation of endogenous dynein IC by CK2. Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with CK2 and [γ- 32 P]ATP. Proteins were resolved and detected as in ( C ). The fold change in phosphorylation was calculated as in ( C ). Data are mean ± range from two independent experiments.

    Journal: Biochemical Journal

    Article Title: Inositol hexakisphosphate kinase 1 (IP6K1) activity is required for cytoplasmic dynein-driven transport

    doi: 10.1042/BCJ20160610

    Figure Lengend Snippet: Inositol pyrophosphates pyrophosphorylate dynein IC. ( A ) Bacterially expressed and purified IC(1–111) was phosphorylated in vitro by CK2, and phosphosite identification was contracted out to the Taplin Mass Spectrometry Facility, Harvard Medical School. The MS/MS spectrum is shown for the doubly phosphorylated peptide corresponding to residues 42–55 of mouse IC-2C (EAAVpSVQEEpSDLEK). The sequence shows the peptide fragmentation pattern, and the table shows masses of all b and y ions, highlighting the ions obtained in the spectrum. Arrows indicate fragment ions containing phosphorylated Ser residues. The mass of fragment y5 indicates phosphorylation of Ser51, and the masses of y10 and b10 correspond to phosphorylation of Ser46 and Ser51. ( B ) Bacterially expressed and purified GST or GST-tagged IC(1–70), IC(1–111), and IC(1–111)S51A were prephosphorylated with CK2 and unlabeled ATP and incubated with 5[β- 32 P]IP 7 . Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and the proteins were detected by Ponceau S staining (left). The phosphorimager scan was subjected to ‘levels’ adjustment in Adobe Photoshop to improve visualization. The image intensity of the pyrophosphorylated protein was normalized to the corresponding total protein. The pyrophosphorylation intensity of each IC fragment was compared with GST. Data are mean ± range from two independent experiments. ( C ) Back-pyrophosphorylation of endogenous dynein IC by IP 7 . Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with 5[β- 32 P]IP 7 , resolved by NuPAGE, and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right), and proteins were detected by western blotting (left). The image intensity of the pyrophosphorylated protein was normalized to the corresponding immunoprecipitated protein. The fold change in the extent of pyrophosphorylation of IC in Ip6k1 −/− compared with Ip6k1 +/+ MEFs is indicated. Data are mean ± SEM from three independent experiments. ( D ) Back-phosphorylation of endogenous dynein IC by CK2. Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with CK2 and [γ- 32 P]ATP. Proteins were resolved and detected as in ( C ). The fold change in phosphorylation was calculated as in ( C ). Data are mean ± range from two independent experiments.

    Article Snippet: Protein on beads was subjected to CK2-mediated phosphorylation by incubating with CK2 enzyme (New England Biolabs) in protein kinase buffer (New England Biolabs) in the presence of 0.5 mM Mg2+ -ATP and 1–2.5 µCi [γ32 -P]ATP for 30 min at 30°C.

    Techniques: Purification, In Vitro, Mass Spectrometry, Sequencing, Incubation, Staining, Immunoprecipitation, Western Blot