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    New England Biolabs xmni
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    Atad5-RLC is the major PCNA unloader in Xenopus egg extracts. A , a schematic diagram of the PCNA unloading assay. Singly biotinylated plasmid carrying a nick was bound to Sepharose beads and incubated with recombinant human PCNA and RFC to load PCNA <t>on</t> <t>DNA.</t> After nick ligation and extensive wash, the resulting PCNA-DNA complex was transferred into Xenopus egg extracts to examine PCNA unloading. B , 0.25 μl each of mock-treated (lane 1), Rfc3-depleted (lane 2), Rfc1-depleted (lane 3), Ctf18-depleted (lane 4), Atad5-depleted (lane 5), Rad17-depleted NPE (lane 6), and a serial dilution series of mock-treated NPE (lanes 7–11) were analyzed by immunoblotting with indicated antibodies. Orc2 serves as a loading control. C , PCNA loaded onto immobilized DNA was incubated in NPE described in ( B ). Untreated DNA separated by agarose gel electrophoresis in the presence of ethidium bromide, a quantitative immunoblot of hPCNA and Rfc3 in the bead-bound fractions, and linearized DNA (treated with <t>XmnI)</t> separated by agarose gel electrophoresis are presented along with the percentage of covalently-closed plasmids, the estimated numbers of DNA-bound PCNA molecules per plasmid, and the amount of bead-bound DNA. D , the average numbers of DNA-bound PCNA per plasmid at the 10-min time points were plotted into a graph. Filled circles represent individual data from two independent experiments, including the one shown in ( C ), except for the ‘Input’ and ‘mock’ samples, which were triplicated.
    Xmni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The Atad5 RFC-like complex is the major unloader of proliferating cell nuclear antigen in Xenopus egg extracts"

    Article Title: The Atad5 RFC-like complex is the major unloader of proliferating cell nuclear antigen in Xenopus egg extracts

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2023.105588

    Atad5-RLC is the major PCNA unloader in Xenopus egg extracts. A , a schematic diagram of the PCNA unloading assay. Singly biotinylated plasmid carrying a nick was bound to Sepharose beads and incubated with recombinant human PCNA and RFC to load PCNA on DNA. After nick ligation and extensive wash, the resulting PCNA-DNA complex was transferred into Xenopus egg extracts to examine PCNA unloading. B , 0.25 μl each of mock-treated (lane 1), Rfc3-depleted (lane 2), Rfc1-depleted (lane 3), Ctf18-depleted (lane 4), Atad5-depleted (lane 5), Rad17-depleted NPE (lane 6), and a serial dilution series of mock-treated NPE (lanes 7–11) were analyzed by immunoblotting with indicated antibodies. Orc2 serves as a loading control. C , PCNA loaded onto immobilized DNA was incubated in NPE described in ( B ). Untreated DNA separated by agarose gel electrophoresis in the presence of ethidium bromide, a quantitative immunoblot of hPCNA and Rfc3 in the bead-bound fractions, and linearized DNA (treated with XmnI) separated by agarose gel electrophoresis are presented along with the percentage of covalently-closed plasmids, the estimated numbers of DNA-bound PCNA molecules per plasmid, and the amount of bead-bound DNA. D , the average numbers of DNA-bound PCNA per plasmid at the 10-min time points were plotted into a graph. Filled circles represent individual data from two independent experiments, including the one shown in ( C ), except for the ‘Input’ and ‘mock’ samples, which were triplicated.
    Figure Legend Snippet: Atad5-RLC is the major PCNA unloader in Xenopus egg extracts. A , a schematic diagram of the PCNA unloading assay. Singly biotinylated plasmid carrying a nick was bound to Sepharose beads and incubated with recombinant human PCNA and RFC to load PCNA on DNA. After nick ligation and extensive wash, the resulting PCNA-DNA complex was transferred into Xenopus egg extracts to examine PCNA unloading. B , 0.25 μl each of mock-treated (lane 1), Rfc3-depleted (lane 2), Rfc1-depleted (lane 3), Ctf18-depleted (lane 4), Atad5-depleted (lane 5), Rad17-depleted NPE (lane 6), and a serial dilution series of mock-treated NPE (lanes 7–11) were analyzed by immunoblotting with indicated antibodies. Orc2 serves as a loading control. C , PCNA loaded onto immobilized DNA was incubated in NPE described in ( B ). Untreated DNA separated by agarose gel electrophoresis in the presence of ethidium bromide, a quantitative immunoblot of hPCNA and Rfc3 in the bead-bound fractions, and linearized DNA (treated with XmnI) separated by agarose gel electrophoresis are presented along with the percentage of covalently-closed plasmids, the estimated numbers of DNA-bound PCNA molecules per plasmid, and the amount of bead-bound DNA. D , the average numbers of DNA-bound PCNA per plasmid at the 10-min time points were plotted into a graph. Filled circles represent individual data from two independent experiments, including the one shown in ( C ), except for the ‘Input’ and ‘mock’ samples, which were triplicated.

    Techniques Used: Plasmid Preparation, Incubation, Recombinant, Ligation, Serial Dilution, Western Blot, Agarose Gel Electrophoresis

    xmni  (New England Biolabs)


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    (A) A schematic diagram of the PCNA unloading assay. Singly biotinylated plasmid carrying a nick was bound to Sepharose beads and incubated with recombinant human PCNA and RFC to load PCNA <t>on</t> <t>DNA.</t> After nick ligation and extensive wash, the resulting PCNA-DNA complex was transferred into Xenopus egg extracts to examine PCNA unloading. (B) 0.25 μL each of mock-treated (lane 1), Rfc3-depleted (lane 2), Rfc1-depleted (lane 3), Ctf18-depleted (lane 4), Atad5-depleted (lane 5), Rad17-depleted NPE (lane 6), and a serial dilution series of mock-treated NPE (lanes 7–11) were analyzed by immunoblotting with indicated antibodies. Orc2 serves as a loading control. (C) PCNA loaded onto immobilized DNA was incubated in NPE described in ( B ). Untreated DNA separated by agarose gel electrophoresis in the presence of ethidium bromide, a quantitative immunoblot of hPCNA and Rfc3 in the bead-bound fractions, and linearized DNA (treated with <t>XmnI)</t> separated by agarose gel electrophoresis are presented along with the percentage of covalently-closed plasmids, the estimated numbers of DNA-bound PCNA molecules per plasmid, and the amount of bead-bound DNA. (D) The average numbers of DNA-bound PCNA per plasmid at the 10-min time points were plotted into a graph. Filled circles represent individual data from two experiments, including the one shown in ( C ).
    Xmni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The Atad5 RFC-like complex is the major unloader of proliferating cell nuclear antigen in Xenopus egg extracts"

    Article Title: The Atad5 RFC-like complex is the major unloader of proliferating cell nuclear antigen in Xenopus egg extracts

    Journal: bioRxiv

    doi: 10.1101/2023.08.28.555229

    (A) A schematic diagram of the PCNA unloading assay. Singly biotinylated plasmid carrying a nick was bound to Sepharose beads and incubated with recombinant human PCNA and RFC to load PCNA on DNA. After nick ligation and extensive wash, the resulting PCNA-DNA complex was transferred into Xenopus egg extracts to examine PCNA unloading. (B) 0.25 μL each of mock-treated (lane 1), Rfc3-depleted (lane 2), Rfc1-depleted (lane 3), Ctf18-depleted (lane 4), Atad5-depleted (lane 5), Rad17-depleted NPE (lane 6), and a serial dilution series of mock-treated NPE (lanes 7–11) were analyzed by immunoblotting with indicated antibodies. Orc2 serves as a loading control. (C) PCNA loaded onto immobilized DNA was incubated in NPE described in ( B ). Untreated DNA separated by agarose gel electrophoresis in the presence of ethidium bromide, a quantitative immunoblot of hPCNA and Rfc3 in the bead-bound fractions, and linearized DNA (treated with XmnI) separated by agarose gel electrophoresis are presented along with the percentage of covalently-closed plasmids, the estimated numbers of DNA-bound PCNA molecules per plasmid, and the amount of bead-bound DNA. (D) The average numbers of DNA-bound PCNA per plasmid at the 10-min time points were plotted into a graph. Filled circles represent individual data from two experiments, including the one shown in ( C ).
    Figure Legend Snippet: (A) A schematic diagram of the PCNA unloading assay. Singly biotinylated plasmid carrying a nick was bound to Sepharose beads and incubated with recombinant human PCNA and RFC to load PCNA on DNA. After nick ligation and extensive wash, the resulting PCNA-DNA complex was transferred into Xenopus egg extracts to examine PCNA unloading. (B) 0.25 μL each of mock-treated (lane 1), Rfc3-depleted (lane 2), Rfc1-depleted (lane 3), Ctf18-depleted (lane 4), Atad5-depleted (lane 5), Rad17-depleted NPE (lane 6), and a serial dilution series of mock-treated NPE (lanes 7–11) were analyzed by immunoblotting with indicated antibodies. Orc2 serves as a loading control. (C) PCNA loaded onto immobilized DNA was incubated in NPE described in ( B ). Untreated DNA separated by agarose gel electrophoresis in the presence of ethidium bromide, a quantitative immunoblot of hPCNA and Rfc3 in the bead-bound fractions, and linearized DNA (treated with XmnI) separated by agarose gel electrophoresis are presented along with the percentage of covalently-closed plasmids, the estimated numbers of DNA-bound PCNA molecules per plasmid, and the amount of bead-bound DNA. (D) The average numbers of DNA-bound PCNA per plasmid at the 10-min time points were plotted into a graph. Filled circles represent individual data from two experiments, including the one shown in ( C ).

    Techniques Used: Plasmid Preparation, Incubation, Recombinant, Ligation, Serial Dilution, Western Blot, Agarose Gel Electrophoresis

    xmni  (New England Biolabs)


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