ni-nta agarose Search Results


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  • 99
    Thermo Fisher ni nta agarose
    The examples of chromatograms of reaction mixture containing initially 80 µM <t>SPI2-SRHWAP-His</t> 6 fusion protein loaded on the <t>Ni-NTA-agarose</t> column, incubated with 4 mM NiCl 2 in 100 mM Hepes buffer, pH 8.2 at 50°C. From top to bottom: control fusion protein (incubated without Ni(II) ions), incubation buffer, and two pooled wash fractions (250 mM imidazole) after 22 h of incubation.
    Ni Nta Agarose, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1834 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen ni nta agarose
    <t>ETV5</t> and TWIST1 interact in vitro and in vivo. ( A ) In vitro pull-down assay showing that recombinant ETV5 can associate with TWIST1 in a cell lysate. Lysate from HEK293T cells transfected with Myc-tagged Twist1 was incubated with either control <t>Ni-NTA</t>
    Ni Nta Agarose, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 30111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen ni nta agarose beads
    Expression of the molecular probe <t>TOP1-DOPA-GFP.</t> (A) A schematic diagram of 30.0 kDa TOP1-DOPA-GFP. (B) Total protein analyses of L-DOPA incorporation into TOP1-DOPA-GFP. TOP1-DOPA-GFP was expressed in the presence and absence of L-DOPA, purified with <t>Ni-NTA</t> resin, and resolved by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (C) Redox cycling staining of TOP1-DOPA-GFP. Proteins from a similar gel as (B) were blotted to a nitrocellulose membrane and stained with NBT reagent (2 M sodium glycinate, 0.24 mM NBT, pH 10). This method detects quino-proteins and confirmed the presence of L-DOPA/dopaquinone in TOP1-DOPA-GFP.
    Ni Nta Agarose Beads, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 13736 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen ni nta magnetic agarose beads
    <t>5e</t> RNA binding activities of polypeptides 72 e, 72 f, 72 g, 72 h and 72 j . Purified his-tagged polypeptides 72 e to 72 j were incubated with in vitro transcribed 5e SRP RNA and <t>Ni-NTA</t> magnetic agarose beads as described in the Methods. The bound protein and RNA were analyzed by SDS PAGE followed by staining of the same gel with Coomassie blue (lanes labeled p) and Ethidium bromide (lanes labeled r). Molecular mass markers in kDa are shown in lane m. Plus signs indicate the formation of complexes, the minus sign below 72 g indicates that this polypeptide was unable to bind. Variable amounts of a material which probably represented 5e SRP RNA dimers were observed (arrow heads).
    Ni Nta Magnetic Agarose Beads, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare ni nta agarose
    Interaction between Pin1 and XPO5 occurs in a phosphorylation-dependent manner. a SK-Hep1 cells were co-transfected with myc-XPO5 and flag-Pin1 or empty vector expression plasmid. After cell lysis, flag-Pin1 was immunoprecipitated with anti-flag antibody. Immunoprecipitates were immunoblotted with anti-myc and anti-flag antibodies. b Lysates from SK-Hep1 cells transfected with myc-XPO5 expression plasmid were incubated with GST or GST-Pin1. GST pull-down complexes were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). c Schematic diagram of full length Pin1 and its mutants (W34A and C113A), Pin1 WW domain, and Pin1 PPIase domain. d Lysates from SK-Hep1 cells transfected with myc-XPO5 expression plasmid were incubated with His-tagged full-length Pin1 (His-Pin1 FL), WW domain (His-Pin1 WW), or PPIase domain (His-Pin1 PPIase), respectively. The pull-down complexes by <t>Ni-NTA</t> agarose beads were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). e SK-Hep1 cells were co-transfected with myc-XPO5 and flag-Pin1 (WT, C113A, or W34A) or empty vector expression plasmid. After cell lysis, flag-Pin1 was immunoprecipitated with anti-flag antibody. Immunoprecipitates were immunoblotted with anti-myc and anti-flag antibodies. f Lysates from SK-Hep1 cells transfected with myc-XPO5 expression plasmid were incubated with or without CIP before the incubation with GST or GST-Pin1. GST pull-down complexes were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). g Lysates from SK-Hep1 cells transfected with myc-XPO5 (WT, 3A, or 3D) or empty vector expression plasmid were incubated with GST or GST-Pin1. GST pull-down complexes were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). 3A: T345A, S416A and S497A mutations; 3D: T345D, S416D and S497D mutations; h Graphic abstract of the mechanism how Pin1 interacts with XPO5
    Ni Nta Agarose, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 953 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher ni nta agarose column
    Interaction between Pin1 and XPO5 occurs in a phosphorylation-dependent manner. a SK-Hep1 cells were co-transfected with myc-XPO5 and flag-Pin1 or empty vector expression plasmid. After cell lysis, flag-Pin1 was immunoprecipitated with anti-flag antibody. Immunoprecipitates were immunoblotted with anti-myc and anti-flag antibodies. b Lysates from SK-Hep1 cells transfected with myc-XPO5 expression plasmid were incubated with GST or GST-Pin1. GST pull-down complexes were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). c Schematic diagram of full length Pin1 and its mutants (W34A and C113A), Pin1 WW domain, and Pin1 PPIase domain. d Lysates from SK-Hep1 cells transfected with myc-XPO5 expression plasmid were incubated with His-tagged full-length Pin1 (His-Pin1 FL), WW domain (His-Pin1 WW), or PPIase domain (His-Pin1 PPIase), respectively. The pull-down complexes by <t>Ni-NTA</t> agarose beads were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). e SK-Hep1 cells were co-transfected with myc-XPO5 and flag-Pin1 (WT, C113A, or W34A) or empty vector expression plasmid. After cell lysis, flag-Pin1 was immunoprecipitated with anti-flag antibody. Immunoprecipitates were immunoblotted with anti-myc and anti-flag antibodies. f Lysates from SK-Hep1 cells transfected with myc-XPO5 expression plasmid were incubated with or without CIP before the incubation with GST or GST-Pin1. GST pull-down complexes were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). g Lysates from SK-Hep1 cells transfected with myc-XPO5 (WT, 3A, or 3D) or empty vector expression plasmid were incubated with GST or GST-Pin1. GST pull-down complexes were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). 3A: T345A, S416A and S497A mutations; 3D: T345D, S416D and S497D mutations; h Graphic abstract of the mechanism how Pin1 interacts with XPO5
    Ni Nta Agarose Column, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL protino ni nta agarose
    Interaction between Pin1 and XPO5 occurs in a phosphorylation-dependent manner. a SK-Hep1 cells were co-transfected with myc-XPO5 and flag-Pin1 or empty vector expression plasmid. After cell lysis, flag-Pin1 was immunoprecipitated with anti-flag antibody. Immunoprecipitates were immunoblotted with anti-myc and anti-flag antibodies. b Lysates from SK-Hep1 cells transfected with myc-XPO5 expression plasmid were incubated with GST or GST-Pin1. GST pull-down complexes were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). c Schematic diagram of full length Pin1 and its mutants (W34A and C113A), Pin1 WW domain, and Pin1 PPIase domain. d Lysates from SK-Hep1 cells transfected with myc-XPO5 expression plasmid were incubated with His-tagged full-length Pin1 (His-Pin1 FL), WW domain (His-Pin1 WW), or PPIase domain (His-Pin1 PPIase), respectively. The pull-down complexes by <t>Ni-NTA</t> agarose beads were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). e SK-Hep1 cells were co-transfected with myc-XPO5 and flag-Pin1 (WT, C113A, or W34A) or empty vector expression plasmid. After cell lysis, flag-Pin1 was immunoprecipitated with anti-flag antibody. Immunoprecipitates were immunoblotted with anti-myc and anti-flag antibodies. f Lysates from SK-Hep1 cells transfected with myc-XPO5 expression plasmid were incubated with or without CIP before the incubation with GST or GST-Pin1. GST pull-down complexes were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). g Lysates from SK-Hep1 cells transfected with myc-XPO5 (WT, 3A, or 3D) or empty vector expression plasmid were incubated with GST or GST-Pin1. GST pull-down complexes were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). 3A: T345A, S416A and S497A mutations; 3D: T345D, S416D and S497D mutations; h Graphic abstract of the mechanism how Pin1 interacts with XPO5
    Protino Ni Nta Agarose, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 88/100, based on 172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The examples of chromatograms of reaction mixture containing initially 80 µM SPI2-SRHWAP-His 6 fusion protein loaded on the Ni-NTA-agarose column, incubated with 4 mM NiCl 2 in 100 mM Hepes buffer, pH 8.2 at 50°C. From top to bottom: control fusion protein (incubated without Ni(II) ions), incubation buffer, and two pooled wash fractions (250 mM imidazole) after 22 h of incubation.

    Journal: PLoS ONE

    Article Title: Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal

    doi: 10.1371/journal.pone.0036350

    Figure Lengend Snippet: The examples of chromatograms of reaction mixture containing initially 80 µM SPI2-SRHWAP-His 6 fusion protein loaded on the Ni-NTA-agarose column, incubated with 4 mM NiCl 2 in 100 mM Hepes buffer, pH 8.2 at 50°C. From top to bottom: control fusion protein (incubated without Ni(II) ions), incubation buffer, and two pooled wash fractions (250 mM imidazole) after 22 h of incubation.

    Article Snippet: On-column affinity tag cleavage The SPI2-SRHWAP-His6 fusion protein was loaded on Ni-NTA-agarose (Invitrogen), according to the manufacturer's instruction.

    Techniques: Incubation

    SUMOylation of human Polλ (A) In vitro SUMOylation assay. Reactions were carried out with purified human Polλ and the indicated purified murine SUMOylation proteins in standard SUMO reaction buffer (see Materials and methods). When indicated, ATP was added to trigger catalytic reactions leading to SUMO conjugation. Proteins were resolved by SDS-PAGE and Polλ was detected by immunoblotting (IB) with anti-Polλ antibodies. In vitro reactions produced ATP-dependent Polλ-SUMO conjugates with slower electrophoretic migration compared with unmodified Polλ. Both unmodified and SUMO-conjugated Polλ are indicated. (B) In vitro SUMOylation assays were performed as in (A), including a GST-SUMO1 fusion protein to confirm Polλ SUMOylation. Proteins were resolved by SDS-PAGE and Polλ was detected by immunoblotting (IB) with anti-Polλ antibodies. The corresponding Polλ-SUMO conjugates with slower electrophoretic migration compared with unmodified Polλ obtained are indicated. GST-SUMO1-Polλ species are in agreement with additional 25 kDa size corresponding to GST protein. (C) In vivo SUMOylation assays. Human 293T cells were transiently transfected with plasmids encoding for Flag-Polλ, His-SUMO1 and Ubc9. SUMO conjugates were purified from cell lysates 48 h later by Ni-NTA pulldown (PD) assays under denaturing conditions. Polλ SUMOylation was detected by immunoblotting (IB) using anti-Flag antibodies. SUMO-Polλ conjugates are observed as slower migrating species compared with unmodified Flag-Polλ. Both unmodified and SUMO-conjugated Polλ are indicated.

    Journal: bioRxiv

    Article Title: RanBP2-mediated SUMOylation promotes human DNA polymerase lambda nuclear localization and DNA repair

    doi: 10.1101/2020.02.14.949644

    Figure Lengend Snippet: SUMOylation of human Polλ (A) In vitro SUMOylation assay. Reactions were carried out with purified human Polλ and the indicated purified murine SUMOylation proteins in standard SUMO reaction buffer (see Materials and methods). When indicated, ATP was added to trigger catalytic reactions leading to SUMO conjugation. Proteins were resolved by SDS-PAGE and Polλ was detected by immunoblotting (IB) with anti-Polλ antibodies. In vitro reactions produced ATP-dependent Polλ-SUMO conjugates with slower electrophoretic migration compared with unmodified Polλ. Both unmodified and SUMO-conjugated Polλ are indicated. (B) In vitro SUMOylation assays were performed as in (A), including a GST-SUMO1 fusion protein to confirm Polλ SUMOylation. Proteins were resolved by SDS-PAGE and Polλ was detected by immunoblotting (IB) with anti-Polλ antibodies. The corresponding Polλ-SUMO conjugates with slower electrophoretic migration compared with unmodified Polλ obtained are indicated. GST-SUMO1-Polλ species are in agreement with additional 25 kDa size corresponding to GST protein. (C) In vivo SUMOylation assays. Human 293T cells were transiently transfected with plasmids encoding for Flag-Polλ, His-SUMO1 and Ubc9. SUMO conjugates were purified from cell lysates 48 h later by Ni-NTA pulldown (PD) assays under denaturing conditions. Polλ SUMOylation was detected by immunoblotting (IB) using anti-Flag antibodies. SUMO-Polλ conjugates are observed as slower migrating species compared with unmodified Flag-Polλ. Both unmodified and SUMO-conjugated Polλ are indicated.

    Article Snippet: Input samples were saved and His-tagged SUMO-conjugated proteins were pulled down from cell extracts by using Ni-NTA agarose beads (Life Technologies).

    Techniques: In Vitro, Purification, Conjugation Assay, SDS Page, Produced, Migration, In Vivo, Transfection

    Physical and functional interaction of RanBP2 and Polλ at the nuclear envelope. (A) Human U2OS cells were transiently co-transfected with a plasmid encoding for HA-RanBP2 together with either Flag-POLL WT or Flag-empty (ev) vectors. Proximity ligation assays (PLA) were performed by using mouse monoclonal anti-RanBP2 and rabbit anti-Polλ antibodies. Subcellular localization of PLA foci (red) was detected by using confocal laser scanning microscopy and nuclei were stained with DAPI (blue). X/Z views are shown in upper and middle panels, with merged images included in the right panels; an X/Y view is also shown in bottom panels, only with Flag-Polλ WT transfected cells. Scale bars, 10 µm. (B) Human 293T cells were transiently transfected as in (A) and HA-RanBP2 was immunoprecipitated 24 h later with anti-HA antibody. Recovered immunocomplexes were analyzed in 4-20% SDS-PAGE and immunoblotting with anti-HA, anti-Flag and anti-tubulin antibodies. (C) Human His-SUMO1 U2OS cells were treated either with control (luciferase) or RanBP2 siRNAs and 24 h later transiently transfected with Flag-POLL WT vector. SUMO-Polλ conjugates were pulled down on Ni-NTA beads under denaturing conditions at indicated times post-transfection and immunoblotted with anti-Flag antibody ( upper panel ). Expression of RanBP2, Flag-Polλ and tubulin was monitored by immunoblotting of cell lysates with the corresponding antibodies (input). (D) Human His-SUMO1 U2OS cells were treated as in (C) and pulled down on Ni-NTA beads under denaturing conditions performed 48 h after vector transfection. Quantification represents percentage of SUMOylated Polλ in cells (mean value ± standard deviation, SD) for each condition analyzed in three independent experiments. Statistical significance was determined by using an unpaired t-test. (E) Human His-SUMO1 U2OS cells were treated as in (C) and subcellular localization of Flag-Polλ was examined by immunofluorescence using anti-Flag antibody (red). Nuclei were stained with DAPI (blue). Representative images of one experiment are shown. Scale bars, 25 µm. Quantification represents percentage of nuclear Polλ in cells (mean value ± standard deviation, SD) for each condition analyzed in three independent experiments, with more than 50 individual cells analyzed for each condition in each independent repeat. Statistical significance was determined by using an unpaired t-test.

    Journal: bioRxiv

    Article Title: RanBP2-mediated SUMOylation promotes human DNA polymerase lambda nuclear localization and DNA repair

    doi: 10.1101/2020.02.14.949644

    Figure Lengend Snippet: Physical and functional interaction of RanBP2 and Polλ at the nuclear envelope. (A) Human U2OS cells were transiently co-transfected with a plasmid encoding for HA-RanBP2 together with either Flag-POLL WT or Flag-empty (ev) vectors. Proximity ligation assays (PLA) were performed by using mouse monoclonal anti-RanBP2 and rabbit anti-Polλ antibodies. Subcellular localization of PLA foci (red) was detected by using confocal laser scanning microscopy and nuclei were stained with DAPI (blue). X/Z views are shown in upper and middle panels, with merged images included in the right panels; an X/Y view is also shown in bottom panels, only with Flag-Polλ WT transfected cells. Scale bars, 10 µm. (B) Human 293T cells were transiently transfected as in (A) and HA-RanBP2 was immunoprecipitated 24 h later with anti-HA antibody. Recovered immunocomplexes were analyzed in 4-20% SDS-PAGE and immunoblotting with anti-HA, anti-Flag and anti-tubulin antibodies. (C) Human His-SUMO1 U2OS cells were treated either with control (luciferase) or RanBP2 siRNAs and 24 h later transiently transfected with Flag-POLL WT vector. SUMO-Polλ conjugates were pulled down on Ni-NTA beads under denaturing conditions at indicated times post-transfection and immunoblotted with anti-Flag antibody ( upper panel ). Expression of RanBP2, Flag-Polλ and tubulin was monitored by immunoblotting of cell lysates with the corresponding antibodies (input). (D) Human His-SUMO1 U2OS cells were treated as in (C) and pulled down on Ni-NTA beads under denaturing conditions performed 48 h after vector transfection. Quantification represents percentage of SUMOylated Polλ in cells (mean value ± standard deviation, SD) for each condition analyzed in three independent experiments. Statistical significance was determined by using an unpaired t-test. (E) Human His-SUMO1 U2OS cells were treated as in (C) and subcellular localization of Flag-Polλ was examined by immunofluorescence using anti-Flag antibody (red). Nuclei were stained with DAPI (blue). Representative images of one experiment are shown. Scale bars, 25 µm. Quantification represents percentage of nuclear Polλ in cells (mean value ± standard deviation, SD) for each condition analyzed in three independent experiments, with more than 50 individual cells analyzed for each condition in each independent repeat. Statistical significance was determined by using an unpaired t-test.

    Article Snippet: Input samples were saved and His-tagged SUMO-conjugated proteins were pulled down from cell extracts by using Ni-NTA agarose beads (Life Technologies).

    Techniques: Functional Assay, Transfection, Plasmid Preparation, Ligation, Proximity Ligation Assay, Confocal Laser Scanning Microscopy, Staining, Immunoprecipitation, SDS Page, Luciferase, Expressing, Standard Deviation, Immunofluorescence

    Increased SUMOylation of Polλ in response to DNA damage. (A) His-SUMO1 and -SUMO2 U2OS cells were grown in p100 dishes until 80% confluence and then harvested. His-SUMO-conjugates were pulled down on Ni-NTA beads (Ni-PD) under denaturing conditions and endogenous SUMO-Polλ was detected by immunoblotting (IB) using anti-Polλ antibody. Expression of Polλ and tubulin was monitored by immunoblotting cell lysates (input) with the corresponding antibodies. (B) His-SUMO2 U2OS cells were grown as described in (A) and either mock-treated or treated with etoposide (20 µM, 30 min), hydrogen peroxide (750 µM, 1 h), MMS (0.03%, 30 min), HU (2 mM, 2h) before harvesting. His-SUMO-conjugates were pulled down on Ni-NTA beads (Ni-PD) and endogenous SUMO-Polλ was detected as described in (A). Expression of Polλ and tubulin was monitored by immunoblotting of cell lysates (input) with the corresponding antibodies. DNA damage induction was confirmed by immunoblotting of cell lysates (input) with anti-phosphorylated H2AX (γH2AX) antibody. ( C ) His-SUMO2 U2OS cells were grown as in (A) and treated with increasing doses of either MMS (0.01%, 0.02% and 0.03%, 30 min) or HU (3 and 5 mM, 2h). His-SUMO-conjugates were detected as described above. Expression of Polλ and tubulin was monitored and replicative stress induction was confirmed with anti-phosphorylated (S4/S8) RPA antibody. ( D ) His-SUMO2 U2OS were synchronized in G1 by using thymidine block, as previously described [ 56 ]. Thymidine block was released from G1-enriched cultures and His-SUMO conjugates were pulled down at different times as indicated in (A). Recovering times were selected according to [ 56 ]. Cell progression at different stages of the cell cycle was monitored by immunoblotting with anti-cyclin A and E antibodies, and expression of Polλ and tubulin was monitored as described before.

    Journal: bioRxiv

    Article Title: RanBP2-mediated SUMOylation promotes human DNA polymerase lambda nuclear localization and DNA repair

    doi: 10.1101/2020.02.14.949644

    Figure Lengend Snippet: Increased SUMOylation of Polλ in response to DNA damage. (A) His-SUMO1 and -SUMO2 U2OS cells were grown in p100 dishes until 80% confluence and then harvested. His-SUMO-conjugates were pulled down on Ni-NTA beads (Ni-PD) under denaturing conditions and endogenous SUMO-Polλ was detected by immunoblotting (IB) using anti-Polλ antibody. Expression of Polλ and tubulin was monitored by immunoblotting cell lysates (input) with the corresponding antibodies. (B) His-SUMO2 U2OS cells were grown as described in (A) and either mock-treated or treated with etoposide (20 µM, 30 min), hydrogen peroxide (750 µM, 1 h), MMS (0.03%, 30 min), HU (2 mM, 2h) before harvesting. His-SUMO-conjugates were pulled down on Ni-NTA beads (Ni-PD) and endogenous SUMO-Polλ was detected as described in (A). Expression of Polλ and tubulin was monitored by immunoblotting of cell lysates (input) with the corresponding antibodies. DNA damage induction was confirmed by immunoblotting of cell lysates (input) with anti-phosphorylated H2AX (γH2AX) antibody. ( C ) His-SUMO2 U2OS cells were grown as in (A) and treated with increasing doses of either MMS (0.01%, 0.02% and 0.03%, 30 min) or HU (3 and 5 mM, 2h). His-SUMO-conjugates were detected as described above. Expression of Polλ and tubulin was monitored and replicative stress induction was confirmed with anti-phosphorylated (S4/S8) RPA antibody. ( D ) His-SUMO2 U2OS were synchronized in G1 by using thymidine block, as previously described [ 56 ]. Thymidine block was released from G1-enriched cultures and His-SUMO conjugates were pulled down at different times as indicated in (A). Recovering times were selected according to [ 56 ]. Cell progression at different stages of the cell cycle was monitored by immunoblotting with anti-cyclin A and E antibodies, and expression of Polλ and tubulin was monitored as described before.

    Article Snippet: Input samples were saved and His-tagged SUMO-conjugated proteins were pulled down from cell extracts by using Ni-NTA agarose beads (Life Technologies).

    Techniques: Expressing, Recombinase Polymerase Amplification, Blocking Assay

    Increased SUMOylation of Polλ in response to HU. His-SUMO1 and -SUMO2 U2OS cells were grown in p100 dishes until 80% confluence, either mock-treated or treated with increasing doses of HU (1 mM, 2 mM and 3 mM) for 2 h, and the harvested. His-SUMO-conjugates were pulled down on Ni-NTA beads (Ni-PD) under denaturing conditions and endogenous SUMO-Polλ was detected by immunoblotting (IB) using anti-Polλ antibody. Expression of Polλ and tubulin was monitored by immunoblotting cell lysates (input) with the corresponding antibodies.

    Journal: bioRxiv

    Article Title: RanBP2-mediated SUMOylation promotes human DNA polymerase lambda nuclear localization and DNA repair

    doi: 10.1101/2020.02.14.949644

    Figure Lengend Snippet: Increased SUMOylation of Polλ in response to HU. His-SUMO1 and -SUMO2 U2OS cells were grown in p100 dishes until 80% confluence, either mock-treated or treated with increasing doses of HU (1 mM, 2 mM and 3 mM) for 2 h, and the harvested. His-SUMO-conjugates were pulled down on Ni-NTA beads (Ni-PD) under denaturing conditions and endogenous SUMO-Polλ was detected by immunoblotting (IB) using anti-Polλ antibody. Expression of Polλ and tubulin was monitored by immunoblotting cell lysates (input) with the corresponding antibodies.

    Article Snippet: Input samples were saved and His-tagged SUMO-conjugated proteins were pulled down from cell extracts by using Ni-NTA agarose beads (Life Technologies).

    Techniques: Expressing

    SUMOylation of human Polλ. (A) In vitro SUMOylation reactions with indicated purified human proteins were carried out as described in Materials and methods. Proteins were resolved by SDS-PAGE and immunoblotted (IB) with anti-Polλ antibody. Unmodified and SUMO-conjugated Polλ are indicated. (B) U2OS cells stably expressing either His-SUMO1 or His-SUMO2 were transiently transfected with empty or Flag-Polλ encoding vectors. His-SUMO-conjugates were pulled down on Ni-NTA beads (Ni-PD) under denaturing conditions (Materials and methods) and immunoblotted (IB) with anti-Flag antibody. Expression of Flag-Polλ was monitored by immunoblotting of cell lysates (input). An U2OS cell line without genomic integration of tagged SUMO proteins was assayed in parallel as a control of specificity. Unmodified and SUMO-conjugated Flag-Polλ are indicated. M denotes the molecular mass markers in kDa (C) Human Polλ domain organization. Main conserved PolX domains [ 42 ] and the localization of putative SUMO acceptor lysines in the N-terminal region are indicated. (D) Identification of SUMO conjugation sites using peptide arrays. Peptides are numbered sequentially from the starting Met (M) codon. Each spot in the array represents Polλ derived peptides formed by the indicated consecutive residues with 10-amino acid overlap with the previous peptide. Polarity of peptides with respect to membrane attachment is indicated with an arrow. K27 is indicated in green and neighbour lysines are marked in bold. The peptide array was subjected to an in vitro SUMOylation assay as described in Materials and methods and then immunoblotted with anti-SUMO1 antibody (right panel). Positive SUMO-conjugated peptides generated dark spots whereas non-conjugated peptides leave blank spots. Left panel represents Ponceau staining of the array. (E, F) U2OS cell lines with integrated His-SUMO1 (E) or His-SUMO2 (F) were transiently transfected either with wild-type Flag-Polλ or Flag-Polλ K27R, 7KR and 6KR mutants. His-SUMO-conjugates were pulled down on Ni-NTA beads (Ni-PD) as in (B) and immunoblotted (IB) with anti-Flag antibody. Expression of Flag-Polλ variants was monitored by immunoblotting of cell lysates (input). Unmodified and SUMO-conjugated Flag-Polλ are indicated.

    Journal: bioRxiv

    Article Title: RanBP2-mediated SUMOylation promotes human DNA polymerase lambda nuclear localization and DNA repair

    doi: 10.1101/2020.02.14.949644

    Figure Lengend Snippet: SUMOylation of human Polλ. (A) In vitro SUMOylation reactions with indicated purified human proteins were carried out as described in Materials and methods. Proteins were resolved by SDS-PAGE and immunoblotted (IB) with anti-Polλ antibody. Unmodified and SUMO-conjugated Polλ are indicated. (B) U2OS cells stably expressing either His-SUMO1 or His-SUMO2 were transiently transfected with empty or Flag-Polλ encoding vectors. His-SUMO-conjugates were pulled down on Ni-NTA beads (Ni-PD) under denaturing conditions (Materials and methods) and immunoblotted (IB) with anti-Flag antibody. Expression of Flag-Polλ was monitored by immunoblotting of cell lysates (input). An U2OS cell line without genomic integration of tagged SUMO proteins was assayed in parallel as a control of specificity. Unmodified and SUMO-conjugated Flag-Polλ are indicated. M denotes the molecular mass markers in kDa (C) Human Polλ domain organization. Main conserved PolX domains [ 42 ] and the localization of putative SUMO acceptor lysines in the N-terminal region are indicated. (D) Identification of SUMO conjugation sites using peptide arrays. Peptides are numbered sequentially from the starting Met (M) codon. Each spot in the array represents Polλ derived peptides formed by the indicated consecutive residues with 10-amino acid overlap with the previous peptide. Polarity of peptides with respect to membrane attachment is indicated with an arrow. K27 is indicated in green and neighbour lysines are marked in bold. The peptide array was subjected to an in vitro SUMOylation assay as described in Materials and methods and then immunoblotted with anti-SUMO1 antibody (right panel). Positive SUMO-conjugated peptides generated dark spots whereas non-conjugated peptides leave blank spots. Left panel represents Ponceau staining of the array. (E, F) U2OS cell lines with integrated His-SUMO1 (E) or His-SUMO2 (F) were transiently transfected either with wild-type Flag-Polλ or Flag-Polλ K27R, 7KR and 6KR mutants. His-SUMO-conjugates were pulled down on Ni-NTA beads (Ni-PD) as in (B) and immunoblotted (IB) with anti-Flag antibody. Expression of Flag-Polλ variants was monitored by immunoblotting of cell lysates (input). Unmodified and SUMO-conjugated Flag-Polλ are indicated.

    Article Snippet: Input samples were saved and His-tagged SUMO-conjugated proteins were pulled down from cell extracts by using Ni-NTA agarose beads (Life Technologies).

    Techniques: In Vitro, Purification, SDS Page, Stable Transfection, Expressing, Transfection, Conjugation Assay, Derivative Assay, Peptide Microarray, Generated, Staining

    Visualization of labeled target complex and enrichment of occupancy The Rpn5 subunit of the wild-type yeast proteasome lid complex was modified by site-directed mutagenesis to contain the amber codon (TAG) at position Y13 (Rpn5 Y13→TAG ) and S26 (Rpn5 S26→TAG ). UAA-containing lid complex (Rpn5 Y13→pAzF or Rpn5 S26→pAzF ) was then generated by in vivo incorporation of the UAA via amber suppression. ( A ). ( B ) Rpn5 secondary structure prediction (ssPRO4.0) places Y13 within a flexible loop, and S26 within an N-terminal alpha-helix. 2D class averages: the left images show unlabeled wild-type and unlabeled Rpn5 Y13→pAzF or Rpn5 S26→pAzF lid particles obtained by negative stain EM. Representative 2D classes from previous 5 . Red arrows indicate electron density corresponding to the MBP label. ( C ) 3D negative stain reconstruction (~20Å resolution) of wild-type lid complex for visual orientation of Rpn5 subunit location within the lid complex. ( D ) Cartoon schematic of a conjugation reaction performed using the GFP Y151→pAzF target reporter protein while bound to its affinity purification resin. Numbers in this panel correspond to the numbered lanes in ( E ) SDS-PAGE analysis of the on-resin conjugation of MBP DBCO (42.5 kDa) to GFP Y151→pAzF (26.9 kDa) with amylose enrichment. A conjugation reaction was performed immediately following capture of 6X His-tagged GFP Y151→pAzF ), 5 ul of resin was used for elution and visualization by SDS gel (lane 1). For the “on-resin” reaction, 50 ul of 250 uM MBP DBCO was added directly to 20 ul of saturated resin, and the reaction was allowed to proceed for 4 hours while turning at 4°C. Ni-NTA resin was then collected, allowing for separation of unreacted MBP DBCO in the flow-through (lane 2) from a resin-bound mixture of conjugated and unconjugated GFP Y151→pAzF reporter protein (eluted and visualized in lane 3). This conjugation reaction was

    Journal: Journal of structural biology

    Article Title: Site-specific labeling of proteins for electron microscopy

    doi: 10.1016/j.jsb.2015.09.010

    Figure Lengend Snippet: Visualization of labeled target complex and enrichment of occupancy The Rpn5 subunit of the wild-type yeast proteasome lid complex was modified by site-directed mutagenesis to contain the amber codon (TAG) at position Y13 (Rpn5 Y13→TAG ) and S26 (Rpn5 S26→TAG ). UAA-containing lid complex (Rpn5 Y13→pAzF or Rpn5 S26→pAzF ) was then generated by in vivo incorporation of the UAA via amber suppression. ( A ). ( B ) Rpn5 secondary structure prediction (ssPRO4.0) places Y13 within a flexible loop, and S26 within an N-terminal alpha-helix. 2D class averages: the left images show unlabeled wild-type and unlabeled Rpn5 Y13→pAzF or Rpn5 S26→pAzF lid particles obtained by negative stain EM. Representative 2D classes from previous 5 . Red arrows indicate electron density corresponding to the MBP label. ( C ) 3D negative stain reconstruction (~20Å resolution) of wild-type lid complex for visual orientation of Rpn5 subunit location within the lid complex. ( D ) Cartoon schematic of a conjugation reaction performed using the GFP Y151→pAzF target reporter protein while bound to its affinity purification resin. Numbers in this panel correspond to the numbered lanes in ( E ) SDS-PAGE analysis of the on-resin conjugation of MBP DBCO (42.5 kDa) to GFP Y151→pAzF (26.9 kDa) with amylose enrichment. A conjugation reaction was performed immediately following capture of 6X His-tagged GFP Y151→pAzF ), 5 ul of resin was used for elution and visualization by SDS gel (lane 1). For the “on-resin” reaction, 50 ul of 250 uM MBP DBCO was added directly to 20 ul of saturated resin, and the reaction was allowed to proceed for 4 hours while turning at 4°C. Ni-NTA resin was then collected, allowing for separation of unreacted MBP DBCO in the flow-through (lane 2) from a resin-bound mixture of conjugated and unconjugated GFP Y151→pAzF reporter protein (eluted and visualized in lane 3). This conjugation reaction was

    Article Snippet: Isolated in the same SEC fractions as the unlabeled lid complexes shown below to the left are: Middle; reference-free 2D class averages of MBP-conjugated lid complex, labeled at amino acid position Y13, and bottom; position S26 in Rpn5 using the maleimide-DBCO reagent shown in reporter protein on Ni-NTA agarose resin.

    Techniques: Labeling, Modification, Mutagenesis, Generated, In Vivo, Staining, Conjugation Assay, Affinity Purification, SDS Page, SDS-Gel, Flow Cytometry

    ETV5 and TWIST1 interact in vitro and in vivo. ( A ) In vitro pull-down assay showing that recombinant ETV5 can associate with TWIST1 in a cell lysate. Lysate from HEK293T cells transfected with Myc-tagged Twist1 was incubated with either control Ni-NTA

    Journal: Development (Cambridge, England)

    Article Title: Preaxial polydactyly: interactions among ETV, TWIST1 and HAND2 control anterior-posterior patterning of the limb

    doi: 10.1242/dev.051789

    Figure Lengend Snippet: ETV5 and TWIST1 interact in vitro and in vivo. ( A ) In vitro pull-down assay showing that recombinant ETV5 can associate with TWIST1 in a cell lysate. Lysate from HEK293T cells transfected with Myc-tagged Twist1 was incubated with either control Ni-NTA

    Article Snippet: His-ETV5 protein was purified by Ni-NTA agarose (Qiagen) from the soluble fraction of the lysate and used to inject rabbits.

    Techniques: In Vitro, In Vivo, Pull Down Assay, Recombinant, Transfection, Incubation

    Purified Syn5 RNAP and transcription on Syn5 DNA. A , SDS-PAGE gel of purified non-tagged Syn5 RNAP ( lane 1 ) and N-terminal His-tagged Syn5 RNAP obtained after a Ni-NTA-agarose column ( lane 2 ), gel filtration column ( lane 3 ), and cellulose phosphate column ( lane 4 ). Purified T7 RNAP is shown for comparison. Proteins were stained with Coomassie Blue. B , 10% denaturing TBE-urea gel of radioactive labeled transcription products generated by Syn5 RNAPs on Syn5 genomic DNA. The lanes in B correspond to transcription by the proteins purified in A .

    Journal: The Journal of Biological Chemistry

    Article Title: The RNA Polymerase of Marine Cyanophage Syn5 *

    doi: 10.1074/jbc.M112.442350

    Figure Lengend Snippet: Purified Syn5 RNAP and transcription on Syn5 DNA. A , SDS-PAGE gel of purified non-tagged Syn5 RNAP ( lane 1 ) and N-terminal His-tagged Syn5 RNAP obtained after a Ni-NTA-agarose column ( lane 2 ), gel filtration column ( lane 3 ), and cellulose phosphate column ( lane 4 ). Purified T7 RNAP is shown for comparison. Proteins were stained with Coomassie Blue. B , 10% denaturing TBE-urea gel of radioactive labeled transcription products generated by Syn5 RNAPs on Syn5 genomic DNA. The lanes in B correspond to transcription by the proteins purified in A .

    Article Snippet: His-tagged Syn5 RNAP was isolated from the lysate using Ni-NTA-agarose chromatography according to the standard Qiagen His-tagged protein purification procedure.

    Techniques: Purification, SDS Page, Filtration, Staining, Labeling, Generated

    Expression of the molecular probe TOP1-DOPA-GFP. (A) A schematic diagram of 30.0 kDa TOP1-DOPA-GFP. (B) Total protein analyses of L-DOPA incorporation into TOP1-DOPA-GFP. TOP1-DOPA-GFP was expressed in the presence and absence of L-DOPA, purified with Ni-NTA resin, and resolved by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (C) Redox cycling staining of TOP1-DOPA-GFP. Proteins from a similar gel as (B) were blotted to a nitrocellulose membrane and stained with NBT reagent (2 M sodium glycinate, 0.24 mM NBT, pH 10). This method detects quino-proteins and confirmed the presence of L-DOPA/dopaquinone in TOP1-DOPA-GFP.

    Journal: Analytical biochemistry

    Article Title: A Versatile Approach to Transform Low-Affinity Peptides into Protein Probes with Co-Translationally Expressed Chemical Cross-Linker 1

    doi: 10.1016/j.ab.2010.05.026

    Figure Lengend Snippet: Expression of the molecular probe TOP1-DOPA-GFP. (A) A schematic diagram of 30.0 kDa TOP1-DOPA-GFP. (B) Total protein analyses of L-DOPA incorporation into TOP1-DOPA-GFP. TOP1-DOPA-GFP was expressed in the presence and absence of L-DOPA, purified with Ni-NTA resin, and resolved by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (C) Redox cycling staining of TOP1-DOPA-GFP. Proteins from a similar gel as (B) were blotted to a nitrocellulose membrane and stained with NBT reagent (2 M sodium glycinate, 0.24 mM NBT, pH 10). This method detects quino-proteins and confirmed the presence of L-DOPA/dopaquinone in TOP1-DOPA-GFP.

    Article Snippet: SH3-His, wtTOP1-GFP, and TOP1-DOPA-GFP were purified using Ni-NTA agarose beads (Qiagen).

    Techniques: Expressing, Purification, SDS Page, Staining

    SDS-PAGE of recombinant FumF protein . Proteins were separated by 12% (w/v) SDS-PAGE and then stained with Coomassie brilliant blue G-250. Lane 1, molecular weight standards; Lane 2, total protein of E. coli BL21(DE3)pLysS harboring empty pETBlue-2 (control); Lane 3, total protein of E. coli BL21(DE3)pLysS harboring the recombinant fumF in pETBlue-2 without induction by IPTG; Lane 4, total protein of E. coli BL21(DE3)pLysS harboring the recombinant fumF in pETBlue-2 induced by addition of 0.5 mM IPTG; Lane 5, sample purified by the Ni-NTA column method. The recombinant FumF protein is indicated by the black arrow.

    Journal: Microbial Cell Factories

    Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

    doi: 10.1186/1475-2859-9-91

    Figure Lengend Snippet: SDS-PAGE of recombinant FumF protein . Proteins were separated by 12% (w/v) SDS-PAGE and then stained with Coomassie brilliant blue G-250. Lane 1, molecular weight standards; Lane 2, total protein of E. coli BL21(DE3)pLysS harboring empty pETBlue-2 (control); Lane 3, total protein of E. coli BL21(DE3)pLysS harboring the recombinant fumF in pETBlue-2 without induction by IPTG; Lane 4, total protein of E. coli BL21(DE3)pLysS harboring the recombinant fumF in pETBlue-2 induced by addition of 0.5 mM IPTG; Lane 5, sample purified by the Ni-NTA column method. The recombinant FumF protein is indicated by the black arrow.

    Article Snippet: The His-tagged FumF protein was expressed and purified using Ni-NTA agarose resin (Qiagen, Valencia, CA, USA), according to the manufacturer's instructions.

    Techniques: SDS Page, Recombinant, Staining, Molecular Weight, Purification

    DNA binding by H115N p53 is greater than that of wild-type p53 as measured by EMSA, filter binding assay, and DNase I footprinting (A) Wild-type p53 and H115N proteins were purified from bacteria by NI-NTA, Heparin and gel-filtration column chromatography as described in Experimental Procedures. The relative amounts of p53 proteins (4, 8, 12 and 16 ng) used in the following assays were detected by immunoblot analysis using mixture of p53 monoclonal antibodies PAb DO-1, 1801 and 421. (B) Wild-type (lanes 2–7) and H115N mutant (lanes 8–13) proteins (4, 8 and 12 ng each) were incubated with a 32 BP DNA duplex containing the 5' p21 promoter p53 binding site (2.5 ng) with PAb 421 (50 ng). DNA was resolved on a 4% polyacrylamide gel, dried and visualized by autoradiography. (C) Either wild-type (solid circles) or H115N (solid squares) p53 protein (each at 0, 10, 20, 30, 40, 50 and 60 ng) was incubated with [ 32 P] labeled DNA duplex (2.5 ng) containing the p21 site and 125 ng of competitor DNA oligonucleotide containing a mutant GADD45 site and then filtered through nitrocellulose membrane. Labeled DNA bound to p53 protein was quantitated by liquid scintilation. (D) Wild-type p53 (100, 300, 500, 700 ng; lanes 3–6) or the same quantities of H115N p53 (lanes 7–10) were incubated with a 5'-end labeled 405 bp HindIII-ScaI fragment (approximately 25 ng) from the p21 promoter containing the 5' p53 binding site and subjected to DNAse I footprinting. Control digestion with DNase I was also performed (lanes 1 and 2, 11 and 12). The p53 binding site is indicated.

    Journal: Cell cycle (Georgetown, Tex.)

    Article Title: Dissection of the sequence-specific DNA binding and exonuclease activities reveals a superactive yet apoptotically impaired mutant p53 protein

    doi:

    Figure Lengend Snippet: DNA binding by H115N p53 is greater than that of wild-type p53 as measured by EMSA, filter binding assay, and DNase I footprinting (A) Wild-type p53 and H115N proteins were purified from bacteria by NI-NTA, Heparin and gel-filtration column chromatography as described in Experimental Procedures. The relative amounts of p53 proteins (4, 8, 12 and 16 ng) used in the following assays were detected by immunoblot analysis using mixture of p53 monoclonal antibodies PAb DO-1, 1801 and 421. (B) Wild-type (lanes 2–7) and H115N mutant (lanes 8–13) proteins (4, 8 and 12 ng each) were incubated with a 32 BP DNA duplex containing the 5' p21 promoter p53 binding site (2.5 ng) with PAb 421 (50 ng). DNA was resolved on a 4% polyacrylamide gel, dried and visualized by autoradiography. (C) Either wild-type (solid circles) or H115N (solid squares) p53 protein (each at 0, 10, 20, 30, 40, 50 and 60 ng) was incubated with [ 32 P] labeled DNA duplex (2.5 ng) containing the p21 site and 125 ng of competitor DNA oligonucleotide containing a mutant GADD45 site and then filtered through nitrocellulose membrane. Labeled DNA bound to p53 protein was quantitated by liquid scintilation. (D) Wild-type p53 (100, 300, 500, 700 ng; lanes 3–6) or the same quantities of H115N p53 (lanes 7–10) were incubated with a 5'-end labeled 405 bp HindIII-ScaI fragment (approximately 25 ng) from the p21 promoter containing the 5' p53 binding site and subjected to DNAse I footprinting. Control digestion with DNase I was also performed (lanes 1 and 2, 11 and 12). The p53 binding site is indicated.

    Article Snippet: Proteins were expressed in BL21(DE3) at 22°C for 2.5 h. The N-terminally His-tagged p53 proteins were first purified on a Ni-NTA agarose column (Qiagen, Valencica, CA).

    Techniques: Binding Assay, Filter-binding Assay, Footprinting, Purification, Filtration, Column Chromatography, Mutagenesis, Incubation, Autoradiography, Labeling

    Phosphorylated C-terminus of Sae2 interacts with Rad50. a Full-length phosphorylated recombinant MBP-tagged pSae2 was mock-treated or dephosphorylated with λ phosphatase upon binding to amylose resin and incubated with recombinant MRX complex. The eluates were visualized by silver staining. Prescission protease was added to all samples as a protein stabilizer and to cleave the MBP tag off pSae2. b The FLAG-tagged recombinant Rad50 protein was immobilized on anti-FLAG affinity resin and incubated with phosphorylated C-terminal domain of pSae2 (pSae2 ΔN169, residues 170–345), which had been either mock-treated or dephosphorylated with λ phosphatase. The bound proteins were eluted and detected by Ponceau staining or western blotting. Avidin was added to elution buffer and shows equal loading. c The phosphorylated recombinant MBP-tagged C-terminal domain of pSae2 (residues 170–345) was bound to amylose resin, eluted, cleaved with prescission protease, and immobilized on NiNTA resin. The bound pSae2 ΔN169 was mock-treated or dephosphorylated with λ phosphatase and incubated with recombinant wild-type Rad50 or ATP binding-deficient Rad50 K40A. Proteins were eluted and visualized by Ponceau staining or western blotting. Avidin was added to elution buffer and shows equal loading. d Assay as in c . Phosphorylated C-terminal domain of pSae2 was incubated with wild-type Rad50 or Rad50 K81I (representative Rad50S) mutant

    Journal: Nature Communications

    Article Title: Regulatory control of DNA end resection by Sae2 phosphorylation

    doi: 10.1038/s41467-018-06417-5

    Figure Lengend Snippet: Phosphorylated C-terminus of Sae2 interacts with Rad50. a Full-length phosphorylated recombinant MBP-tagged pSae2 was mock-treated or dephosphorylated with λ phosphatase upon binding to amylose resin and incubated with recombinant MRX complex. The eluates were visualized by silver staining. Prescission protease was added to all samples as a protein stabilizer and to cleave the MBP tag off pSae2. b The FLAG-tagged recombinant Rad50 protein was immobilized on anti-FLAG affinity resin and incubated with phosphorylated C-terminal domain of pSae2 (pSae2 ΔN169, residues 170–345), which had been either mock-treated or dephosphorylated with λ phosphatase. The bound proteins were eluted and detected by Ponceau staining or western blotting. Avidin was added to elution buffer and shows equal loading. c The phosphorylated recombinant MBP-tagged C-terminal domain of pSae2 (residues 170–345) was bound to amylose resin, eluted, cleaved with prescission protease, and immobilized on NiNTA resin. The bound pSae2 ΔN169 was mock-treated or dephosphorylated with λ phosphatase and incubated with recombinant wild-type Rad50 or ATP binding-deficient Rad50 K40A. Proteins were eluted and visualized by Ponceau staining or western blotting. Avidin was added to elution buffer and shows equal loading. d Assay as in c . Phosphorylated C-terminal domain of pSae2 was incubated with wild-type Rad50 or Rad50 K81I (representative Rad50S) mutant

    Article Snippet: Finally, pSae2 was purified using NiNTA agarose (Qiagen).

    Techniques: Recombinant, Binding Assay, Incubation, Silver Staining, Staining, Western Blot, Avidin-Biotin Assay, Mutagenesis

    Chemical cross-linking of in vitro–translated HA1-RAE1 to E . coli –purified GLEBS-like motifs. (A) Purified recombinant HIS-NUP98(150–224) (∼11-kD) protein separated by SDS-PAGE (15% polyacrylamide gel) and detected by Coomassie brilliant blue (CBB; Bio-Rad Laboratories) staining. (B) Purified recombinant GST (29 kD) and GST-NUP98(150–224) (∼38 kD) protein run on a 10% polyacrylamide gel and stained with CBB. (C) Pull-down assays performed with [ 35 S]-methionine–labeled HA1-RAE1 synthesized in vitro (45 kD), and GST- or HIS-NUP98(150–224) fusion proteins purified from E . coli . 5% input shows 5% of the labeled HA1-RAE1 protein used in each pull-down assay. Typically, the in vitro–translated RAE1 appears as a doublet, representing fragments with and without a HA1 tag (the RAE1 cDNA was cloned in pSP73 as a HA1 fusion gene that retained the endogenous RAE1 translation initiation codon). GST beads and Ni-NTA agarose acted as negative control for binding in GST-NUP98(150–224) and HIS-NUP98(150–224) pull-down assays, respectively. Comparable amounts of GST, GST-NUP98(150–224), and HIS-NUP98(150–224) proteins were used in each pull-down assay. The experiment shown is representative for two independent experiments. A cross-linked GST-NUP98(150–224)/RAE1 product of ∼84 kD and a cross-linked HIS-NUP98(150–224)/RAE1 product of ∼56 kD were obtained specifically in DSS-treated samples. Note that cross-linking of RAE1 to HIS-NUP98(150–224) was more efficient than to GST-NUP98(150–224). Molecular weight standards are indicated at right.

    Journal: The Journal of Cell Biology

    Article Title: RAE1 Is a Shuttling mRNA Export Factor That Binds to a GLEBS-like NUP98 Motif at the Nuclear Pore Complex through Multiple Domains

    doi:

    Figure Lengend Snippet: Chemical cross-linking of in vitro–translated HA1-RAE1 to E . coli –purified GLEBS-like motifs. (A) Purified recombinant HIS-NUP98(150–224) (∼11-kD) protein separated by SDS-PAGE (15% polyacrylamide gel) and detected by Coomassie brilliant blue (CBB; Bio-Rad Laboratories) staining. (B) Purified recombinant GST (29 kD) and GST-NUP98(150–224) (∼38 kD) protein run on a 10% polyacrylamide gel and stained with CBB. (C) Pull-down assays performed with [ 35 S]-methionine–labeled HA1-RAE1 synthesized in vitro (45 kD), and GST- or HIS-NUP98(150–224) fusion proteins purified from E . coli . 5% input shows 5% of the labeled HA1-RAE1 protein used in each pull-down assay. Typically, the in vitro–translated RAE1 appears as a doublet, representing fragments with and without a HA1 tag (the RAE1 cDNA was cloned in pSP73 as a HA1 fusion gene that retained the endogenous RAE1 translation initiation codon). GST beads and Ni-NTA agarose acted as negative control for binding in GST-NUP98(150–224) and HIS-NUP98(150–224) pull-down assays, respectively. Comparable amounts of GST, GST-NUP98(150–224), and HIS-NUP98(150–224) proteins were used in each pull-down assay. The experiment shown is representative for two independent experiments. A cross-linked GST-NUP98(150–224)/RAE1 product of ∼84 kD and a cross-linked HIS-NUP98(150–224)/RAE1 product of ∼56 kD were obtained specifically in DSS-treated samples. Note that cross-linking of RAE1 to HIS-NUP98(150–224) was more efficient than to GST-NUP98(150–224). Molecular weight standards are indicated at right.

    Article Snippet: HIS-tagged recombinant mouse RAE1 protein was produced in Escherichia coli DH12S cells, purified with Ni-NTA (nitrilotriacetic acid) agarose beads (QIAGEN Inc.) according to the manufacturer's instructions, and injected into rabbits.

    Techniques: In Vitro, Purification, Recombinant, SDS Page, Staining, Labeling, Synthesized, Pull Down Assay, Clone Assay, Negative Control, Binding Assay, Molecular Weight

    Proteasomal ATPases associate into a heteromeric complex. His 6 -Rpt1 was expressed in a Δ rpt1 background (DY19). Extracts from His 6 -Rpt1-expressing and wild-type (WT) control strains were partially purified on DEAE–CL-6B resin in the absence of ATP. The 500 mM NaCl eluate was fractionated on Ni-NTA affinity columns. Column fractions were subjected to immunoblotting (A) and tested for peptidase activity against Suc-LLVY-AMC (B). The epitope-tagged complex eluting at 100 mM imidazole contained a number of RP subunits (Rpt1, Rpt6, and Rpn10) (A) but lacked peptidase activity (B). The wild-type complex eluted during low-imidazole rinses. (C) Extracts from strains expressing His 6 -tagged versions of each of the six ATPases were also purified by Ni-NTA chromatography. Fractions loaded onto the Ni-NTA column (Load) were compared to fractions from the 100 mM imidazole eluate (Eluate) by immunoblotting with anti-Rpt1 and anti-Rpt6 antibodies.

    Journal: Molecular and Cellular Biology

    Article Title: The Regulatory Particle of the Saccharomyces cerevisiae Proteasome

    doi:

    Figure Lengend Snippet: Proteasomal ATPases associate into a heteromeric complex. His 6 -Rpt1 was expressed in a Δ rpt1 background (DY19). Extracts from His 6 -Rpt1-expressing and wild-type (WT) control strains were partially purified on DEAE–CL-6B resin in the absence of ATP. The 500 mM NaCl eluate was fractionated on Ni-NTA affinity columns. Column fractions were subjected to immunoblotting (A) and tested for peptidase activity against Suc-LLVY-AMC (B). The epitope-tagged complex eluting at 100 mM imidazole contained a number of RP subunits (Rpt1, Rpt6, and Rpn10) (A) but lacked peptidase activity (B). The wild-type complex eluted during low-imidazole rinses. (C) Extracts from strains expressing His 6 -tagged versions of each of the six ATPases were also purified by Ni-NTA chromatography. Fractions loaded onto the Ni-NTA column (Load) were compared to fractions from the 100 mM imidazole eluate (Eluate) by immunoblotting with anti-Rpt1 and anti-Rpt6 antibodies.

    Article Snippet: Briefly, eluates from the DEAE–Affi-Gel Blue column were loaded onto Ni-NTA–agarose columns (Qiagen).

    Techniques: Expressing, Purification, Activity Assay, Chromatography

    The proteasome is a heteromeric complex of ATPases. His 6 -Rpt2 was expressed in a Δ rpt2 background (DY17). Extracts from His 6 -Rpt2-expressing and wild-type (WT) control strains were partially purified by DEAE–Affi-Gel Blue chromatography in the presence of 1 mM Mg-ATP. The 150 mM NaCl eluate was subjected to Ni-NTA affinity chromatography. Column fractions were immunoblotted (A) and tested for peptidase activity against Suc-LLVY-AMC (B). The epitope-tagged complex eluted at 100 mM imidazole, as indicated by immunoblotting against Rpt1, Rpt6, and Rpn10 (A) and by peptidase activity (B). The wild-type proteasome eluted during low-imidazole rinses. (C) Extracts from strains expressing His 6 -tagged versions of each of the six ATPases were individually purified by Ni-NTA chromatography. Fractions loaded onto the Ni-NTA column (Load) were compared to fractions from the 100 mM imidazole eluate (Eluate) by immunoblotting with anti-Rpt1 and anti-Rpt6 antibodies.

    Journal: Molecular and Cellular Biology

    Article Title: The Regulatory Particle of the Saccharomyces cerevisiae Proteasome

    doi:

    Figure Lengend Snippet: The proteasome is a heteromeric complex of ATPases. His 6 -Rpt2 was expressed in a Δ rpt2 background (DY17). Extracts from His 6 -Rpt2-expressing and wild-type (WT) control strains were partially purified by DEAE–Affi-Gel Blue chromatography in the presence of 1 mM Mg-ATP. The 150 mM NaCl eluate was subjected to Ni-NTA affinity chromatography. Column fractions were immunoblotted (A) and tested for peptidase activity against Suc-LLVY-AMC (B). The epitope-tagged complex eluted at 100 mM imidazole, as indicated by immunoblotting against Rpt1, Rpt6, and Rpn10 (A) and by peptidase activity (B). The wild-type proteasome eluted during low-imidazole rinses. (C) Extracts from strains expressing His 6 -tagged versions of each of the six ATPases were individually purified by Ni-NTA chromatography. Fractions loaded onto the Ni-NTA column (Load) were compared to fractions from the 100 mM imidazole eluate (Eluate) by immunoblotting with anti-Rpt1 and anti-Rpt6 antibodies.

    Article Snippet: Briefly, eluates from the DEAE–Affi-Gel Blue column were loaded onto Ni-NTA–agarose columns (Qiagen).

    Techniques: Expressing, Purification, Chromatography, Affinity Column, Activity Assay

    5e RNA binding activities of polypeptides 72 e, 72 f, 72 g, 72 h and 72 j . Purified his-tagged polypeptides 72 e to 72 j were incubated with in vitro transcribed 5e SRP RNA and Ni-NTA magnetic agarose beads as described in the Methods. The bound protein and RNA were analyzed by SDS PAGE followed by staining of the same gel with Coomassie blue (lanes labeled p) and Ethidium bromide (lanes labeled r). Molecular mass markers in kDa are shown in lane m. Plus signs indicate the formation of complexes, the minus sign below 72 g indicates that this polypeptide was unable to bind. Variable amounts of a material which probably represented 5e SRP RNA dimers were observed (arrow heads).

    Journal: BMC Molecular Biology

    Article Title: Identification of amino acid residues in protein SRP72 required for binding to a kinked 5e motif of the human signal recognition particle RNA

    doi: 10.1186/1471-2199-11-83

    Figure Lengend Snippet: 5e RNA binding activities of polypeptides 72 e, 72 f, 72 g, 72 h and 72 j . Purified his-tagged polypeptides 72 e to 72 j were incubated with in vitro transcribed 5e SRP RNA and Ni-NTA magnetic agarose beads as described in the Methods. The bound protein and RNA were analyzed by SDS PAGE followed by staining of the same gel with Coomassie blue (lanes labeled p) and Ethidium bromide (lanes labeled r). Molecular mass markers in kDa are shown in lane m. Plus signs indicate the formation of complexes, the minus sign below 72 g indicates that this polypeptide was unable to bind. Variable amounts of a material which probably represented 5e SRP RNA dimers were observed (arrow heads).

    Article Snippet: Magnetic bead binding assays Binding of the his-tagged polypeptides to the 5e or Δ35 SRP RNA was measured using Ni-NTA magnetic agarose beads (Quiagen) essentially as described [ ].

    Techniques: RNA Binding Assay, Purification, Incubation, In Vitro, SDS Page, Staining, Labeling

    5e SRP RNA binding activities of mutated SRP72 fragments . For each construct, 82.5 ng of in vitro transcribed 5e SRP RNA was incubated with purified protein at the concentrations 0.056, 0.18, 0.56, and 1.8 uM. Samples were incubated with Ni-NTA magnetic agarose beads as described in the Methods. The bound protein and RNA were analyzed by SDS PAGE followed by staining with Coomassie blue (labeled p below each panel) and Ethidium bromide. The amount of bound RNA was measured and normalized to 100 percent in reference to the binding observed with 72 h, 72 j, or 72 k. The insert on the bottom right depicts the design of the assay.

    Journal: BMC Molecular Biology

    Article Title: Identification of amino acid residues in protein SRP72 required for binding to a kinked 5e motif of the human signal recognition particle RNA

    doi: 10.1186/1471-2199-11-83

    Figure Lengend Snippet: 5e SRP RNA binding activities of mutated SRP72 fragments . For each construct, 82.5 ng of in vitro transcribed 5e SRP RNA was incubated with purified protein at the concentrations 0.056, 0.18, 0.56, and 1.8 uM. Samples were incubated with Ni-NTA magnetic agarose beads as described in the Methods. The bound protein and RNA were analyzed by SDS PAGE followed by staining with Coomassie blue (labeled p below each panel) and Ethidium bromide. The amount of bound RNA was measured and normalized to 100 percent in reference to the binding observed with 72 h, 72 j, or 72 k. The insert on the bottom right depicts the design of the assay.

    Article Snippet: Magnetic bead binding assays Binding of the his-tagged polypeptides to the 5e or Δ35 SRP RNA was measured using Ni-NTA magnetic agarose beads (Quiagen) essentially as described [ ].

    Techniques: RNA Binding Assay, Construct, In Vitro, Incubation, Purification, SDS Page, Staining, Labeling, Binding Assay

    Chymotryptic protection in the RNA binding domain of SRP72 . a. Digestion of purified 72 j (lane 1) with Chymotrypsin for 3, 6 or 10 minutes (lanes 2 to 4). Similarly, digestion of purified 72 k (lane 5) for 3, 6 or 10 minutes (lanes 6 to 8). Lane 9, the 72 l fragment (lacking residues 584 to 590) treated with Chymotrypsin for 10 minutes; lane 10, the 585/6 polypeptide, generated by a 10 minute chymotryptic digest of 72 k, retained on Ni-NTA magnetic agarose beads (see Methods for details). All samples were analyzed by electrophoresis on 12.5 percent SDS polyacrylamide gels followed by staining of the polypeptides with Coomassie blue. Molecular mass markers in kDa are indicated on the left and the migrations of Chymotrypsin (CT) and the his-tagged C-terminal 585/6 fragment are marked on the right. b. Digestion of 1.2 μg 72 j with Chymotrypsin in the absence of RNA (lane 1) or in the presence of increasing amounts of human Δ35 RNA (lanes 2 to 4)[ 23 ]. Indicated are molecular mass markers and the migrations of Chymotrypsin (CT), the 72 j polypeptide, and the his-tagged oligopeptide generated by the cleavages of the Tyrosines 585 or 586. c. Binding of human Δ35 to increasing amounts of 72 j. d. Results from gelshift experiment for the binding data shown in Figure 5c. Indicated are the mobilities of the free Δ35 RNA (fr) and its complex with 72 j (cp). Lane numbers correspond to the seven data points shown in Figure 5c. e. Similar to Figure 5 d, showing the formation of a complex between the 72 j and the 5e SRP RNA.

    Journal: BMC Molecular Biology

    Article Title: Identification of amino acid residues in protein SRP72 required for binding to a kinked 5e motif of the human signal recognition particle RNA

    doi: 10.1186/1471-2199-11-83

    Figure Lengend Snippet: Chymotryptic protection in the RNA binding domain of SRP72 . a. Digestion of purified 72 j (lane 1) with Chymotrypsin for 3, 6 or 10 minutes (lanes 2 to 4). Similarly, digestion of purified 72 k (lane 5) for 3, 6 or 10 minutes (lanes 6 to 8). Lane 9, the 72 l fragment (lacking residues 584 to 590) treated with Chymotrypsin for 10 minutes; lane 10, the 585/6 polypeptide, generated by a 10 minute chymotryptic digest of 72 k, retained on Ni-NTA magnetic agarose beads (see Methods for details). All samples were analyzed by electrophoresis on 12.5 percent SDS polyacrylamide gels followed by staining of the polypeptides with Coomassie blue. Molecular mass markers in kDa are indicated on the left and the migrations of Chymotrypsin (CT) and the his-tagged C-terminal 585/6 fragment are marked on the right. b. Digestion of 1.2 μg 72 j with Chymotrypsin in the absence of RNA (lane 1) or in the presence of increasing amounts of human Δ35 RNA (lanes 2 to 4)[ 23 ]. Indicated are molecular mass markers and the migrations of Chymotrypsin (CT), the 72 j polypeptide, and the his-tagged oligopeptide generated by the cleavages of the Tyrosines 585 or 586. c. Binding of human Δ35 to increasing amounts of 72 j. d. Results from gelshift experiment for the binding data shown in Figure 5c. Indicated are the mobilities of the free Δ35 RNA (fr) and its complex with 72 j (cp). Lane numbers correspond to the seven data points shown in Figure 5c. e. Similar to Figure 5 d, showing the formation of a complex between the 72 j and the 5e SRP RNA.

    Article Snippet: Magnetic bead binding assays Binding of the his-tagged polypeptides to the 5e or Δ35 SRP RNA was measured using Ni-NTA magnetic agarose beads (Quiagen) essentially as described [ ].

    Techniques: RNA Binding Assay, Purification, Generated, Electrophoresis, Staining, Binding Assay

    SsoRad54 protein interacts directly with SsoRadA protein. ( A ) Purified SsoRad54 protein was incubated with or without SsoRadA protein prior to the addition of Ni-NTA magnetic beads. The beads were magnetically sequestered and subjected to extensive washes to remove unbound proteins. Control reactions in lanes 2 and 3 represent the binding of SsoRad54 or SsoRadA, respectively, to the magnetic beads; lanes 5 and 6 show SsoRad54 and SsoRadA present in the supernatant following magnetic sequestration of the beads. Lane 1 shows the interaction between SsoRad54 and SsoRadA as revealed by binding of the protein complex to the beads, while lane 4 shows the protein that remains unbound and free in the supernatant. ( B ) SsoRad54 also interacts with Thermus thermophilus RecA but not with Thermotoga maritima LDH ( C ) or SsoSSB ( D ). Migration position for each protein is indicated to the right of each gel.

    Journal: Nucleic Acids Research

    Article Title: An archaeal Rad54 protein remodels DNA and stimulates DNA strand exchange by RadA

    doi: 10.1093/nar/gkp068

    Figure Lengend Snippet: SsoRad54 protein interacts directly with SsoRadA protein. ( A ) Purified SsoRad54 protein was incubated with or without SsoRadA protein prior to the addition of Ni-NTA magnetic beads. The beads were magnetically sequestered and subjected to extensive washes to remove unbound proteins. Control reactions in lanes 2 and 3 represent the binding of SsoRad54 or SsoRadA, respectively, to the magnetic beads; lanes 5 and 6 show SsoRad54 and SsoRadA present in the supernatant following magnetic sequestration of the beads. Lane 1 shows the interaction between SsoRad54 and SsoRadA as revealed by binding of the protein complex to the beads, while lane 4 shows the protein that remains unbound and free in the supernatant. ( B ) SsoRad54 also interacts with Thermus thermophilus RecA but not with Thermotoga maritima LDH ( C ) or SsoSSB ( D ). Migration position for each protein is indicated to the right of each gel.

    Article Snippet: Reaction mixtures contained interaction buffer [30 mM MES (pH 6.5), 200 mM NaCl, 15 mM Mg(OAc)2 , 50 mM imidazole and 0.2% Triton X-100], 0.06 μM SsoRad54, and 0.2 μM SsoRadA, Thermus thermophilus RecA (NEB), Thermotoga maritima LDH, or SsoSSB as indicated and were incubated at 80°C for 1 h. Ni-NTA magnetic beads were then added to a final concentration of 1% and incubation was continued at 25°C for 1 h. Beads were separated from the solution phase by a QIAGEN ‘12-Tube Magnet’ and were washed with 2 × 500 μl of interaction buffer to remove free proteins.

    Techniques: Purification, Incubation, Magnetic Beads, Binding Assay, Migration

    Interaction between Pin1 and XPO5 occurs in a phosphorylation-dependent manner. a SK-Hep1 cells were co-transfected with myc-XPO5 and flag-Pin1 or empty vector expression plasmid. After cell lysis, flag-Pin1 was immunoprecipitated with anti-flag antibody. Immunoprecipitates were immunoblotted with anti-myc and anti-flag antibodies. b Lysates from SK-Hep1 cells transfected with myc-XPO5 expression plasmid were incubated with GST or GST-Pin1. GST pull-down complexes were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). c Schematic diagram of full length Pin1 and its mutants (W34A and C113A), Pin1 WW domain, and Pin1 PPIase domain. d Lysates from SK-Hep1 cells transfected with myc-XPO5 expression plasmid were incubated with His-tagged full-length Pin1 (His-Pin1 FL), WW domain (His-Pin1 WW), or PPIase domain (His-Pin1 PPIase), respectively. The pull-down complexes by Ni-NTA agarose beads were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). e SK-Hep1 cells were co-transfected with myc-XPO5 and flag-Pin1 (WT, C113A, or W34A) or empty vector expression plasmid. After cell lysis, flag-Pin1 was immunoprecipitated with anti-flag antibody. Immunoprecipitates were immunoblotted with anti-myc and anti-flag antibodies. f Lysates from SK-Hep1 cells transfected with myc-XPO5 expression plasmid were incubated with or without CIP before the incubation with GST or GST-Pin1. GST pull-down complexes were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). g Lysates from SK-Hep1 cells transfected with myc-XPO5 (WT, 3A, or 3D) or empty vector expression plasmid were incubated with GST or GST-Pin1. GST pull-down complexes were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). 3A: T345A, S416A and S497A mutations; 3D: T345D, S416D and S497D mutations; h Graphic abstract of the mechanism how Pin1 interacts with XPO5

    Journal: Cell Death and Differentiation

    Article Title: Pin1 impairs microRNA biogenesis by mediating conformation change of XPO5 in hepatocellular carcinoma

    doi: 10.1038/s41418-018-0065-z

    Figure Lengend Snippet: Interaction between Pin1 and XPO5 occurs in a phosphorylation-dependent manner. a SK-Hep1 cells were co-transfected with myc-XPO5 and flag-Pin1 or empty vector expression plasmid. After cell lysis, flag-Pin1 was immunoprecipitated with anti-flag antibody. Immunoprecipitates were immunoblotted with anti-myc and anti-flag antibodies. b Lysates from SK-Hep1 cells transfected with myc-XPO5 expression plasmid were incubated with GST or GST-Pin1. GST pull-down complexes were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). c Schematic diagram of full length Pin1 and its mutants (W34A and C113A), Pin1 WW domain, and Pin1 PPIase domain. d Lysates from SK-Hep1 cells transfected with myc-XPO5 expression plasmid were incubated with His-tagged full-length Pin1 (His-Pin1 FL), WW domain (His-Pin1 WW), or PPIase domain (His-Pin1 PPIase), respectively. The pull-down complexes by Ni-NTA agarose beads were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). e SK-Hep1 cells were co-transfected with myc-XPO5 and flag-Pin1 (WT, C113A, or W34A) or empty vector expression plasmid. After cell lysis, flag-Pin1 was immunoprecipitated with anti-flag antibody. Immunoprecipitates were immunoblotted with anti-myc and anti-flag antibodies. f Lysates from SK-Hep1 cells transfected with myc-XPO5 expression plasmid were incubated with or without CIP before the incubation with GST or GST-Pin1. GST pull-down complexes were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). g Lysates from SK-Hep1 cells transfected with myc-XPO5 (WT, 3A, or 3D) or empty vector expression plasmid were incubated with GST or GST-Pin1. GST pull-down complexes were subjected to SDS-PAGE and immunoblotted with anti-myc antibody or stained by Coomassie blue (CB). 3A: T345A, S416A and S497A mutations; 3D: T345D, S416D and S497D mutations; h Graphic abstract of the mechanism how Pin1 interacts with XPO5

    Article Snippet: Glutathione-Sepharose and Ni-NTA agarose were obtained from GE Healthcare (USA).

    Techniques: Transfection, Plasmid Preparation, Expressing, Lysis, Immunoprecipitation, Incubation, SDS Page, Staining

    BCCIPβ interacts with human RAD51. ( A ) Human RAD51 (5 μg) was incubated with Ni-NTA beads in the presence (lanes 1–3) or absence of BCCIPβ-(HIS) 6 (5 μg; lanes 4–6). ( B ) Sc Rad51 (5 μg) was incubated with Ni-NTA in the absence (lanes 1–3) or presence of BCCIPβ-(HIS) 6 (5 μg; lanes 4–6). The supernatant was removed, the beads were washed, and the bound proteins were eluted with SDS. The supernatant (S), wash (W) and eluate (E), were resolved using SDS-PAGE and stained with Coomassie blue.

    Journal: Nucleic Acids Research

    Article Title: The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing

    doi: 10.1093/nar/gkw877

    Figure Lengend Snippet: BCCIPβ interacts with human RAD51. ( A ) Human RAD51 (5 μg) was incubated with Ni-NTA beads in the presence (lanes 1–3) or absence of BCCIPβ-(HIS) 6 (5 μg; lanes 4–6). ( B ) Sc Rad51 (5 μg) was incubated with Ni-NTA in the absence (lanes 1–3) or presence of BCCIPβ-(HIS) 6 (5 μg; lanes 4–6). The supernatant was removed, the beads were washed, and the bound proteins were eluted with SDS. The supernatant (S), wash (W) and eluate (E), were resolved using SDS-PAGE and stained with Coomassie blue.

    Article Snippet: As a control, RAD51 (5 μg) or Sc Rad51 (5 μg) was incubated with Ni-NTA agarose beads in the absence of BCCIPβ-(HIS)6 , under the same experimental conditions as above.

    Techniques: Incubation, SDS Page, Staining

    Dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified CsnB. M, molecular marker; 1, crude enzymes; 2, purified CsnB. The molecular weight of purified CsnB (theoretical molecular: 30.89 kDa) after Ni-NTA sepharose column was measured to be about 30 kDa by SDS-PAGE.

    Journal: Molecules

    Article Title: Cloning and Characterization of a Cold-adapted Chitosanase from Marine Bacterium Bacillus sp. BY01

    doi: 10.3390/molecules24213915

    Figure Lengend Snippet: Dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified CsnB. M, molecular marker; 1, crude enzymes; 2, purified CsnB. The molecular weight of purified CsnB (theoretical molecular: 30.89 kDa) after Ni-NTA sepharose column was measured to be about 30 kDa by SDS-PAGE.

    Article Snippet: Purification and Characterization of csnB After centrifuging the cultures at 4 °C with 9000 rpm for 20 min, the supernatant was loaded onto a Ni-NTA sepharose column (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and performed purification process with AKTA150 automatic purification system.

    Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Marker, Molecular Weight