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After addition of 2 μl of 50% glycerol, the reaction mixtures were directly applied on a polyacrylamide gel containing 6 mM magnesium acetate and 5% glycerol in 0.5× TBE, and electrophoresis was conducted at room temperature. For DNA helicase assays, the reaction mixtures of the gel shift assays, as described above, were incubated at 30°C for 15 min, and then the ATP was added at 10 mM, followed by incubation at 37°C for additional 30 min. The reactions were terminated by addition of EDTA (20 mM), SDS (0.1%) and 2 μg proteinase K, and were incubated for additional 15 min. The samples were separated by electrophoresis on nondenaturing polyacrylamide gel in 1× TBE."}

{"project":"craft-ca-core-ex-dev","denotations":[{"id":"T2602","span":{"begin":0,"end":3},"obj":"CHEBI_SO_EXT:DNA"},{"id":"T2603","span":{"begin":0,"end":12},"obj":"GO_EXT:0003678"},{"id":"T2604","span":{"begin":55,"end":59},"obj":"PR_EXT:000010246"},{"id":"T2605","span":{"begin":55,"end":63},"obj":"GO:0097373"},{"id":"T2606","span":{"begin":64,"end":72},"obj":"CHEBI_PR_EXT:protein"},{"id":"T2607","span":{"begin":119,"end":127},"obj":"CHEBI:60004"},{"id":"T2608","span":{"begin":153,"end":158},"obj":"CHEBI:46756"},{"id":"T2609","span":{"begin":159,"end":163},"obj":"CHEBI:32145"},{"id":"T2610","span":{"begin":180,"end":194},"obj":"CHEBI:32954"},{"id":"T2611","span":{"begin":202,"end":213},"obj":"CHEBI:62964"},{"id":"T2612","span":{"begin":220,"end":223},"obj":"CHEBI:18320"},{"id":"T2613","span":{"begin":248,"end":255},"obj":"CHEBI:27575"},{"id":"T2614","span":{"begin":260,"end":267},"obj":"CHEBI_SO_EXT:molecular_label_or_mark_or_tag_process"},{"id":"T2615","span":{"begin":334,"end":342},"obj":"CHEBI:17754"},{"id":"T2616","span":{"begin":357,"end":365},"obj":"CHEBI:60004"},{"id":"T2617","span":{"begin":393,"end":407},"obj":"CHEBI_EXT:polyacrylamide"},{"id":"T2618","span":{"begin":428,"end":445},"obj":"CHEBI:62964"},{"id":"T2619","span":{"begin":453,"end":461},"obj":"CHEBI:17754"},{"id":"T2620","span":{"begin":534,"end":537},"obj":"CHEBI_SO_EXT:DNA"},{"id":"T2621","span":{"begin":534,"end":546},"obj":"GO_EXT:0003678"},{"id":"T2622","span":{"begin":568,"end":576},"obj":"CHEBI:60004"},{"id":"T2623","span":{"begin":670,"end":673},"obj":"CHEBI_EXT:ATP"},{"id":"T2624","span":{"begin":793,"end":797},"obj":"CHEBI_EXT:EDTA"},{"id":"T2625","span":{"begin":807,"end":810},"obj":"CHEBI:8984"},{"id":"T2626","span":{"begin":827,"end":837},"obj":"GO_EXT:0008233"},{"id":"T2627","span":{"begin":935,"end":945},"obj":"GO_EXT:macromolecule_denaturation"},{"id":"T2628","span":{"begin":946,"end":960},"obj":"CHEBI_EXT:polyacrylamide"}],"text":"DNA helicase and gel shift assays\nIn gel shift assays, Mcm4/6/7 proteins were incubated at 30°C for 30 min in reaction mixtures (15 μl) containing 25 mM HEPES–NaOH (pH 7.5), 50 mM sodium acetate, 10 mM Mg(CH3COO)2, 1 mM DTT, 0.25 μg/ml BSA, 0.5 mM ATP-γ-S and labeled substrates of the amount indicated. After addition of 2 μl of 50% glycerol, the reaction mixtures were directly applied on a polyacrylamide gel containing 6 mM magnesium acetate and 5% glycerol in 0.5× TBE, and electrophoresis was conducted at room temperature. For DNA helicase assays, the reaction mixtures of the gel shift assays, as described above, were incubated at 30°C for 15 min, and then the ATP was added at 10 mM, followed by incubation at 37°C for additional 30 min. The reactions were terminated by addition of EDTA (20 mM), SDS (0.1%) and 2 μg proteinase K, and were incubated for additional 15 min. The samples were separated by electrophoresis on nondenaturing polyacrylamide gel in 1× TBE."}

{"project":"craft-ca-core-dev","denotations":[{"id":"T2587","span":{"begin":55,"end":59},"obj":"PR:000010246"},{"id":"T2588","span":{"begin":55,"end":63},"obj":"GO:0097373"},{"id":"T2589","span":{"begin":119,"end":127},"obj":"CHEBI:60004"},{"id":"T2590","span":{"begin":153,"end":158},"obj":"CHEBI:46756"},{"id":"T2591","span":{"begin":159,"end":163},"obj":"CHEBI:32145"},{"id":"T2592","span":{"begin":180,"end":194},"obj":"CHEBI:32954"},{"id":"T2593","span":{"begin":202,"end":213},"obj":"CHEBI:62964"},{"id":"T2594","span":{"begin":220,"end":223},"obj":"CHEBI:18320"},{"id":"T2595","span":{"begin":248,"end":255},"obj":"CHEBI:27575"},{"id":"T2596","span":{"begin":334,"end":342},"obj":"CHEBI:17754"},{"id":"T2597","span":{"begin":357,"end":365},"obj":"CHEBI:60004"},{"id":"T2598","span":{"begin":428,"end":445},"obj":"CHEBI:62964"},{"id":"T2599","span":{"begin":453,"end":461},"obj":"CHEBI:17754"},{"id":"T2600","span":{"begin":568,"end":576},"obj":"CHEBI:60004"},{"id":"T2601","span":{"begin":807,"end":810},"obj":"CHEBI:8984"}],"text":"DNA helicase and gel shift assays\nIn gel shift assays, Mcm4/6/7 proteins were incubated at 30°C for 30 min in reaction mixtures (15 μl) containing 25 mM HEPES–NaOH (pH 7.5), 50 mM sodium acetate, 10 mM Mg(CH3COO)2, 1 mM DTT, 0.25 μg/ml BSA, 0.5 mM ATP-γ-S and labeled substrates of the amount indicated. After addition of 2 μl of 50% glycerol, the reaction mixtures were directly applied on a polyacrylamide gel containing 6 mM magnesium acetate and 5% glycerol in 0.5× TBE, and electrophoresis was conducted at room temperature. For DNA helicase assays, the reaction mixtures of the gel shift assays, as described above, were incubated at 30°C for 15 min, and then the ATP was added at 10 mM, followed by incubation at 37°C for additional 30 min. The reactions were terminated by addition of EDTA (20 mM), SDS (0.1%) and 2 μg proteinase K, and were incubated for additional 15 min. The samples were separated by electrophoresis on nondenaturing polyacrylamide gel in 1× TBE."}