u recombinant apex1  (New England Biolabs)


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    Structured Review

    New England Biolabs u recombinant apex1
    NEIL1 and NEIL2 are required for removal of 5fC and 5caC in HeLa cells. ( a ) Scheme of DNA glycosylase assay. A 160 bp synthetic, fluorescently-labeled oligonucleotide containing the indicated cytosine derivative was incubated for N-glycosidic bond cleavage by HeLa extract DNA glycosylases in absence of MgCl 2 . Base excision generates an abasic site, which can be converted into a single-strand break (nucleotide position 79) by recombinant <t>APEX1,</t> and monitored by denaturing PAGE. ( b ) Electropherograms of DNA glycosylase assays. Product peaks of glycosylase activities (79mers; arrows) are highlighted red with efficiencies shown as % of 79 nt peak integral relative to the total fluorescent signal per electropherogram. Data are representative of three independent experiments. ( c ) siRNA screen for DNA glycosylases required for 5fC and 5caC removal. Data represent percentage of abasic sites (% AP site) generated during the assay as quantified from strand cleavage products (79mer) post APEX1 treatment. ( d ) DNA glycosylase assay using HeLa extracts depleted of TDG and NEIL1 + NEIL2 by siRNAs. ( e ) DNA demodification assay as in Figure 1 b but with HeLa cell extracts depleted as in d . ( f ) LC-MS/MS quantification of genomic cytosine modifications from HeLa cells siRNA depleted of the indicated genes. Cells were transfected with TET1 catalytic domain to elevate levels of rare demethylation intermediates and to facilitate their mass-spectrometric detection. Error bars, s.d. (n = 3 assay repetitions ( c-e ) or cell culture transfections ( f )). n.s., not significant. * P
    U Recombinant Apex1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 82/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u recombinant apex1/product/New England Biolabs
    Average 82 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    u recombinant apex1 - by Bioz Stars, 2019-07
    82/100 stars

    Images

    1) Product Images from "Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation"

    Article Title: Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.3151

    NEIL1 and NEIL2 are required for removal of 5fC and 5caC in HeLa cells. ( a ) Scheme of DNA glycosylase assay. A 160 bp synthetic, fluorescently-labeled oligonucleotide containing the indicated cytosine derivative was incubated for N-glycosidic bond cleavage by HeLa extract DNA glycosylases in absence of MgCl 2 . Base excision generates an abasic site, which can be converted into a single-strand break (nucleotide position 79) by recombinant APEX1, and monitored by denaturing PAGE. ( b ) Electropherograms of DNA glycosylase assays. Product peaks of glycosylase activities (79mers; arrows) are highlighted red with efficiencies shown as % of 79 nt peak integral relative to the total fluorescent signal per electropherogram. Data are representative of three independent experiments. ( c ) siRNA screen for DNA glycosylases required for 5fC and 5caC removal. Data represent percentage of abasic sites (% AP site) generated during the assay as quantified from strand cleavage products (79mer) post APEX1 treatment. ( d ) DNA glycosylase assay using HeLa extracts depleted of TDG and NEIL1 + NEIL2 by siRNAs. ( e ) DNA demodification assay as in Figure 1 b but with HeLa cell extracts depleted as in d . ( f ) LC-MS/MS quantification of genomic cytosine modifications from HeLa cells siRNA depleted of the indicated genes. Cells were transfected with TET1 catalytic domain to elevate levels of rare demethylation intermediates and to facilitate their mass-spectrometric detection. Error bars, s.d. (n = 3 assay repetitions ( c-e ) or cell culture transfections ( f )). n.s., not significant. * P
    Figure Legend Snippet: NEIL1 and NEIL2 are required for removal of 5fC and 5caC in HeLa cells. ( a ) Scheme of DNA glycosylase assay. A 160 bp synthetic, fluorescently-labeled oligonucleotide containing the indicated cytosine derivative was incubated for N-glycosidic bond cleavage by HeLa extract DNA glycosylases in absence of MgCl 2 . Base excision generates an abasic site, which can be converted into a single-strand break (nucleotide position 79) by recombinant APEX1, and monitored by denaturing PAGE. ( b ) Electropherograms of DNA glycosylase assays. Product peaks of glycosylase activities (79mers; arrows) are highlighted red with efficiencies shown as % of 79 nt peak integral relative to the total fluorescent signal per electropherogram. Data are representative of three independent experiments. ( c ) siRNA screen for DNA glycosylases required for 5fC and 5caC removal. Data represent percentage of abasic sites (% AP site) generated during the assay as quantified from strand cleavage products (79mer) post APEX1 treatment. ( d ) DNA glycosylase assay using HeLa extracts depleted of TDG and NEIL1 + NEIL2 by siRNAs. ( e ) DNA demodification assay as in Figure 1 b but with HeLa cell extracts depleted as in d . ( f ) LC-MS/MS quantification of genomic cytosine modifications from HeLa cells siRNA depleted of the indicated genes. Cells were transfected with TET1 catalytic domain to elevate levels of rare demethylation intermediates and to facilitate their mass-spectrometric detection. Error bars, s.d. (n = 3 assay repetitions ( c-e ) or cell culture transfections ( f )). n.s., not significant. * P

    Techniques Used: Labeling, Incubation, Recombinant, Polyacrylamide Gel Electrophoresis, Generated, Liquid Chromatography, Mass Spectrometry, Transfection, Cell Culture

    Related Articles

    Nucleic Acid Electrophoresis:

    Article Title: Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation
    Article Snippet: To generate strand breaks at AP sites reaction products were incubated with 5 u recombinant APEX1 (M0282, NEB) for 30 min at 37 °C in a total volume of 10 µl. .. To generate strand breaks at AP sites reaction products were incubated with 5 u recombinant APEX1 (M0282, NEB) for 30 min at 37 °C in a total volume of 10 µl.

    Transfection:

    Article Title: Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation
    Article Snippet: HeLa cells were harvested 48h after siRNA transfection and whole cell extracts were prepared as described above. .. To generate strand breaks at AP sites reaction products were incubated with 5 u recombinant APEX1 (M0282, NEB) for 30 min at 37 °C in a total volume of 10 µl.

    Isolation:

    Article Title: Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation
    Article Snippet: Reactions were stopped and DNA was isolated as described above. .. To generate strand breaks at AP sites reaction products were incubated with 5 u recombinant APEX1 (M0282, NEB) for 30 min at 37 °C in a total volume of 10 µl.

    Electrophoresis:

    Article Title: Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation
    Article Snippet: To generate strand breaks at AP sites reaction products were incubated with 5 u recombinant APEX1 (M0282, NEB) for 30 min at 37 °C in a total volume of 10 µl. .. Enzymatic strand cleavage by APEX1 instead of alkali treatment of the reaction products proved necessary since DNA substrates containing 5-formyl- or 5-carboxylcytidine residues showed significant strand cleavage after boiling in 150 mM NaOH even in absence of any prior enzymatic incubation (data not shown).

    Incubation:

    Article Title: Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation
    Article Snippet: Reactions were stopped and DNA was isolated as described above. .. To generate strand breaks at AP sites reaction products were incubated with 5 u recombinant APEX1 (M0282, NEB) for 30 min at 37 °C in a total volume of 10 µl. .. Enzymatic strand cleavage by APEX1 instead of alkali treatment of the reaction products proved necessary since DNA substrates containing 5-formyl- or 5-carboxylcytidine residues showed significant strand cleavage after boiling in 150 mM NaOH even in absence of any prior enzymatic incubation (data not shown).

    Recombinant:

    Article Title: Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation
    Article Snippet: Reactions were stopped and DNA was isolated as described above. .. To generate strand breaks at AP sites reaction products were incubated with 5 u recombinant APEX1 (M0282, NEB) for 30 min at 37 °C in a total volume of 10 µl. .. Enzymatic strand cleavage by APEX1 instead of alkali treatment of the reaction products proved necessary since DNA substrates containing 5-formyl- or 5-carboxylcytidine residues showed significant strand cleavage after boiling in 150 mM NaOH even in absence of any prior enzymatic incubation (data not shown).

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    New England Biolabs u recombinant apex1
    NEIL1 and NEIL2 are required for removal of 5fC and 5caC in HeLa cells. ( a ) Scheme of DNA glycosylase assay. A 160 bp synthetic, fluorescently-labeled oligonucleotide containing the indicated cytosine derivative was incubated for N-glycosidic bond cleavage by HeLa extract DNA glycosylases in absence of MgCl 2 . Base excision generates an abasic site, which can be converted into a single-strand break (nucleotide position 79) by recombinant <t>APEX1,</t> and monitored by denaturing PAGE. ( b ) Electropherograms of DNA glycosylase assays. Product peaks of glycosylase activities (79mers; arrows) are highlighted red with efficiencies shown as % of 79 nt peak integral relative to the total fluorescent signal per electropherogram. Data are representative of three independent experiments. ( c ) siRNA screen for DNA glycosylases required for 5fC and 5caC removal. Data represent percentage of abasic sites (% AP site) generated during the assay as quantified from strand cleavage products (79mer) post APEX1 treatment. ( d ) DNA glycosylase assay using HeLa extracts depleted of TDG and NEIL1 + NEIL2 by siRNAs. ( e ) DNA demodification assay as in Figure 1 b but with HeLa cell extracts depleted as in d . ( f ) LC-MS/MS quantification of genomic cytosine modifications from HeLa cells siRNA depleted of the indicated genes. Cells were transfected with TET1 catalytic domain to elevate levels of rare demethylation intermediates and to facilitate their mass-spectrometric detection. Error bars, s.d. (n = 3 assay repetitions ( c-e ) or cell culture transfections ( f )). n.s., not significant. * P
    U Recombinant Apex1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 82/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u recombinant apex1/product/New England Biolabs
    Average 82 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    u recombinant apex1 - by Bioz Stars, 2019-07
    82/100 stars
      Buy from Supplier

    Image Search Results


    NEIL1 and NEIL2 are required for removal of 5fC and 5caC in HeLa cells. ( a ) Scheme of DNA glycosylase assay. A 160 bp synthetic, fluorescently-labeled oligonucleotide containing the indicated cytosine derivative was incubated for N-glycosidic bond cleavage by HeLa extract DNA glycosylases in absence of MgCl 2 . Base excision generates an abasic site, which can be converted into a single-strand break (nucleotide position 79) by recombinant APEX1, and monitored by denaturing PAGE. ( b ) Electropherograms of DNA glycosylase assays. Product peaks of glycosylase activities (79mers; arrows) are highlighted red with efficiencies shown as % of 79 nt peak integral relative to the total fluorescent signal per electropherogram. Data are representative of three independent experiments. ( c ) siRNA screen for DNA glycosylases required for 5fC and 5caC removal. Data represent percentage of abasic sites (% AP site) generated during the assay as quantified from strand cleavage products (79mer) post APEX1 treatment. ( d ) DNA glycosylase assay using HeLa extracts depleted of TDG and NEIL1 + NEIL2 by siRNAs. ( e ) DNA demodification assay as in Figure 1 b but with HeLa cell extracts depleted as in d . ( f ) LC-MS/MS quantification of genomic cytosine modifications from HeLa cells siRNA depleted of the indicated genes. Cells were transfected with TET1 catalytic domain to elevate levels of rare demethylation intermediates and to facilitate their mass-spectrometric detection. Error bars, s.d. (n = 3 assay repetitions ( c-e ) or cell culture transfections ( f )). n.s., not significant. * P

    Journal: Nature structural & molecular biology

    Article Title: Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation

    doi: 10.1038/nsmb.3151

    Figure Lengend Snippet: NEIL1 and NEIL2 are required for removal of 5fC and 5caC in HeLa cells. ( a ) Scheme of DNA glycosylase assay. A 160 bp synthetic, fluorescently-labeled oligonucleotide containing the indicated cytosine derivative was incubated for N-glycosidic bond cleavage by HeLa extract DNA glycosylases in absence of MgCl 2 . Base excision generates an abasic site, which can be converted into a single-strand break (nucleotide position 79) by recombinant APEX1, and monitored by denaturing PAGE. ( b ) Electropherograms of DNA glycosylase assays. Product peaks of glycosylase activities (79mers; arrows) are highlighted red with efficiencies shown as % of 79 nt peak integral relative to the total fluorescent signal per electropherogram. Data are representative of three independent experiments. ( c ) siRNA screen for DNA glycosylases required for 5fC and 5caC removal. Data represent percentage of abasic sites (% AP site) generated during the assay as quantified from strand cleavage products (79mer) post APEX1 treatment. ( d ) DNA glycosylase assay using HeLa extracts depleted of TDG and NEIL1 + NEIL2 by siRNAs. ( e ) DNA demodification assay as in Figure 1 b but with HeLa cell extracts depleted as in d . ( f ) LC-MS/MS quantification of genomic cytosine modifications from HeLa cells siRNA depleted of the indicated genes. Cells were transfected with TET1 catalytic domain to elevate levels of rare demethylation intermediates and to facilitate their mass-spectrometric detection. Error bars, s.d. (n = 3 assay repetitions ( c-e ) or cell culture transfections ( f )). n.s., not significant. * P

    Article Snippet: To generate strand breaks at AP sites reaction products were incubated with 5 u recombinant APEX1 (M0282, NEB) for 30 min at 37 °C in a total volume of 10 µl.

    Techniques: Labeling, Incubation, Recombinant, Polyacrylamide Gel Electrophoresis, Generated, Liquid Chromatography, Mass Spectrometry, Transfection, Cell Culture