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  • 99
    Thermo Fisher atp determination kit
    Atp Determination Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 1 article reviews
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    atp determination kit - by Bioz Stars, 2021-03
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    97
    Millipore mgatp
    Mgatp, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mgatp - by Bioz Stars, 2021-03
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    90
    PerkinElmer γ 32 p atp
    γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/PerkinElmer
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    86
    GE Healthcare γ 32 p atp
    Ubiquitin is a direct substrate of TcPINK1 in vitro ( A ) Sequence alignment of residues around Ser 65 in human ubiquitin and a range of ubiquitin-like modifiers and Ubl-domain-containing human proteins. ( B ) Ubiquitin and Parkin are specific substrates of TcPINK1. The indicated ubiquitin-like modifiers and Ubl-domain-containing proteins (1 μg) were incubated with wild-type (WT) or kinase-inactive (KI) TcPINK1 and Mg 2+ -[γ- 32 <t>P]ATP</t> for 60 min. Assays were terminated by the addition of SDS loading buffer and products were analysed by SDS/PAGE. Proteins were detected by Colloidal Coomassie Blue staining (top panel) and incorporation of [γ- 32 P]ATP was detected by autoradiography (bottom panel). All substrates were of human sequence and expressed in E. coli . Tags on the substrates used for this experiment were GST–OTU1, untagged Nedd8, untagged ISG15, His 6 –SUMO1-(1–97), ubiquilin2 (His 6 –SUMO tag cleaved off), His 6 –HOIL1 and USP4. Asterisks denote the correct substrate band. ( C ) TcPINK1 is a specific upstream kinase of ubiquitin. The indicated kinases (1 μg) were incubated with ubiquitin and Mg 2+ -[γ- 32 P]ATP for 60 min. Assays were terminated and analysed as described in ( B ). Except for PINK1, all kinases were of human sequence and expression tags used were GST–MLK1-(132–413), GST–CDK2-(2–298)/cyclin A2-(171–432); His 6 –IKKε-(1–716), His 6 –IKKβ-(1–716), His 6 –Aurora A-(2–403), His 6 –NUAK1-(2–660), His 6 –GSK3β-(2–420) and His 6 –PLK1-(1–603). Broken dividing lines indicate separate gels; asterisks denote the kinase band. The molecular mass in kDa is indicated.
    γ 32 P Atp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/GE Healthcare
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    γ 32 p atp - by Bioz Stars, 2021-03
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    N/A
    Assay Kit for detection of ATP in the research laboratory
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    Image Search Results


    Ubiquitin is a direct substrate of TcPINK1 in vitro ( A ) Sequence alignment of residues around Ser 65 in human ubiquitin and a range of ubiquitin-like modifiers and Ubl-domain-containing human proteins. ( B ) Ubiquitin and Parkin are specific substrates of TcPINK1. The indicated ubiquitin-like modifiers and Ubl-domain-containing proteins (1 μg) were incubated with wild-type (WT) or kinase-inactive (KI) TcPINK1 and Mg 2+ -[γ- 32 P]ATP for 60 min. Assays were terminated by the addition of SDS loading buffer and products were analysed by SDS/PAGE. Proteins were detected by Colloidal Coomassie Blue staining (top panel) and incorporation of [γ- 32 P]ATP was detected by autoradiography (bottom panel). All substrates were of human sequence and expressed in E. coli . Tags on the substrates used for this experiment were GST–OTU1, untagged Nedd8, untagged ISG15, His 6 –SUMO1-(1–97), ubiquilin2 (His 6 –SUMO tag cleaved off), His 6 –HOIL1 and USP4. Asterisks denote the correct substrate band. ( C ) TcPINK1 is a specific upstream kinase of ubiquitin. The indicated kinases (1 μg) were incubated with ubiquitin and Mg 2+ -[γ- 32 P]ATP for 60 min. Assays were terminated and analysed as described in ( B ). Except for PINK1, all kinases were of human sequence and expression tags used were GST–MLK1-(132–413), GST–CDK2-(2–298)/cyclin A2-(171–432); His 6 –IKKε-(1–716), His 6 –IKKβ-(1–716), His 6 –Aurora A-(2–403), His 6 –NUAK1-(2–660), His 6 –GSK3β-(2–420) and His 6 –PLK1-(1–603). Broken dividing lines indicate separate gels; asterisks denote the kinase band. The molecular mass in kDa is indicated.

    Journal: Biochemical Journal

    Article Title: Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65

    doi: 10.1042/BJ20140334

    Figure Lengend Snippet: Ubiquitin is a direct substrate of TcPINK1 in vitro ( A ) Sequence alignment of residues around Ser 65 in human ubiquitin and a range of ubiquitin-like modifiers and Ubl-domain-containing human proteins. ( B ) Ubiquitin and Parkin are specific substrates of TcPINK1. The indicated ubiquitin-like modifiers and Ubl-domain-containing proteins (1 μg) were incubated with wild-type (WT) or kinase-inactive (KI) TcPINK1 and Mg 2+ -[γ- 32 P]ATP for 60 min. Assays were terminated by the addition of SDS loading buffer and products were analysed by SDS/PAGE. Proteins were detected by Colloidal Coomassie Blue staining (top panel) and incorporation of [γ- 32 P]ATP was detected by autoradiography (bottom panel). All substrates were of human sequence and expressed in E. coli . Tags on the substrates used for this experiment were GST–OTU1, untagged Nedd8, untagged ISG15, His 6 –SUMO1-(1–97), ubiquilin2 (His 6 –SUMO tag cleaved off), His 6 –HOIL1 and USP4. Asterisks denote the correct substrate band. ( C ) TcPINK1 is a specific upstream kinase of ubiquitin. The indicated kinases (1 μg) were incubated with ubiquitin and Mg 2+ -[γ- 32 P]ATP for 60 min. Assays were terminated and analysed as described in ( B ). Except for PINK1, all kinases were of human sequence and expression tags used were GST–MLK1-(132–413), GST–CDK2-(2–298)/cyclin A2-(171–432); His 6 –IKKε-(1–716), His 6 –IKKβ-(1–716), His 6 –Aurora A-(2–403), His 6 –NUAK1-(2–660), His 6 –GSK3β-(2–420) and His 6 –PLK1-(1–603). Broken dividing lines indicate separate gels; asterisks denote the kinase band. The molecular mass in kDa is indicated.

    Article Snippet: Incorporation of [γ-32 P]ATP into substrates was analysed by autoradiography using Amersham Hyperfilm.

    Techniques: In Vitro, Sequencing, Incubation, SDS Page, Staining, Autoradiography, Expressing

    TcPINK1 phosphorylates ubiquitin at Ser 65 ( A ) Mapping of phosphopeptide on ubiquitin after phosphorylation by TcPINK1 in vitro . Ubiquitin (10 μg) was incubated with 10 μg of either wild-type TcPINK1 or kinase-inactive TcPINK1 (D359A) in the presence of Mg 2+ -[γ- 32 P]ATP for 80 min. Assays were terminated by the addition of SDS loading buffer and products were separated by SDS/PAGE. Proteins were detected by Colloidal Coomassie Blue staining and phosphorylated ubiquitin was digested with trypsin. Peptides were chromatographed on a reverse-phase HPLC Vydac C 18 column equilibrated in 0.1% trifluoroacetic acid and the column developed with a linear acetonitrile gradient at a flow rate of 0.2 ml/min and fractions (0.1 ml each) were collected and analysed for 32 P radioactivity by Cerenkov counting. One major 32 P-labelled peak was identified following incubation with wild-type TcPINK1, whereas no peaks were identified following incubation with kinase-inactive TcPINK1 (results not shown). ( B ) The phosphopeptide identified in ( A ) was analysed by solid-phase Edman sequencing and MS. The amino acid sequence deduced from the single phosphopeptide seen in the LC–MS/MS analysis is shown using the single-letter amino acid code. ( C ) The S65A mutation abolishes ubiquitin phosphorylation by TcPINK1. Wild-type or S65A mutant ubiquitin (1 μg) was incubated in the presence of wild-type or kinase-inactive TcPINK1 (1 μg) and Mg 2+ -[γ- 32 P]ATP for the times indicated and assays were terminated by the addition of SDS loading buffer. Samples were subjected to SDS/PAGE and proteins detected by Colloidal Coomassie Blue staining (bottom panels) and incorporation of [γ- 32 P]ATP was detected by autoradiography (top panels). Cerenkov counting was used to calculate the stoichiometry of ubiquitin phosphorylation indicated above autoradiographs as mol of [γ- 32 P]ATP incorporated/mol of ubiquitin. KI, kinase-inactive; WT, wild-type.

    Journal: Biochemical Journal

    Article Title: Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65

    doi: 10.1042/BJ20140334

    Figure Lengend Snippet: TcPINK1 phosphorylates ubiquitin at Ser 65 ( A ) Mapping of phosphopeptide on ubiquitin after phosphorylation by TcPINK1 in vitro . Ubiquitin (10 μg) was incubated with 10 μg of either wild-type TcPINK1 or kinase-inactive TcPINK1 (D359A) in the presence of Mg 2+ -[γ- 32 P]ATP for 80 min. Assays were terminated by the addition of SDS loading buffer and products were separated by SDS/PAGE. Proteins were detected by Colloidal Coomassie Blue staining and phosphorylated ubiquitin was digested with trypsin. Peptides were chromatographed on a reverse-phase HPLC Vydac C 18 column equilibrated in 0.1% trifluoroacetic acid and the column developed with a linear acetonitrile gradient at a flow rate of 0.2 ml/min and fractions (0.1 ml each) were collected and analysed for 32 P radioactivity by Cerenkov counting. One major 32 P-labelled peak was identified following incubation with wild-type TcPINK1, whereas no peaks were identified following incubation with kinase-inactive TcPINK1 (results not shown). ( B ) The phosphopeptide identified in ( A ) was analysed by solid-phase Edman sequencing and MS. The amino acid sequence deduced from the single phosphopeptide seen in the LC–MS/MS analysis is shown using the single-letter amino acid code. ( C ) The S65A mutation abolishes ubiquitin phosphorylation by TcPINK1. Wild-type or S65A mutant ubiquitin (1 μg) was incubated in the presence of wild-type or kinase-inactive TcPINK1 (1 μg) and Mg 2+ -[γ- 32 P]ATP for the times indicated and assays were terminated by the addition of SDS loading buffer. Samples were subjected to SDS/PAGE and proteins detected by Colloidal Coomassie Blue staining (bottom panels) and incorporation of [γ- 32 P]ATP was detected by autoradiography (top panels). Cerenkov counting was used to calculate the stoichiometry of ubiquitin phosphorylation indicated above autoradiographs as mol of [γ- 32 P]ATP incorporated/mol of ubiquitin. KI, kinase-inactive; WT, wild-type.

    Article Snippet: Incorporation of [γ-32 P]ATP into substrates was analysed by autoradiography using Amersham Hyperfilm.

    Techniques: In Vitro, Incubation, SDS Page, Staining, High Performance Liquid Chromatography, Flow Cytometry, Radioactivity, Sequencing, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Autoradiography

    PINK1-dependent phosphorylation of ubiquitin at Ser 65 leads to increased activity of ΔUbl-Parkin ( A ) MBP–TcPINK1 enhances ΔUbl-Parkin-mediated ubiquitin discharge from UbcH7. ΔUbl-Parkin was phosphorylated using wild-type (WT), kinase-inactive (KI) or no MBP–TcPINK1 in the presence of Mg 2+ -[γ- 32 P]ATP. An E2-discharge assay was established by incubation of this mixture with 2 μg of UbcH7 that had been pre-incubated with E1 and FLAG–ubiquitin in the presence of ATP for 60 min. Reactions were allowed to continue for 15 min and were stopped using SDS loading buffer in the absence of reducing agent. Samples were analysed as described in the Materials and methods section. Protein phosphorylation was monitored by autoradiography (bottom panel). ( B ) ΔUbl-Parkin activity is increased by wild-type TcPINK1. A 2 μg amount of full-length (WT) or ΔUbl-Parkin was incubated with 1 μg of wild-type (WT), kinase-inactive (KI) or no TcPINK1 in a kinase reaction for 60 min. The ubiquitylation reactions were then initiated by the addition of ubiquitylation assay components (E1, UbcH7 and FLAG–ubiquitin) and 2 μg of His 6 –SUMO-Miro1. Reactions were terminated after 60 min by the addition of SDS loading buffer and products were analysed by SDS/PAGE. Miro1, ubiquitin (Ub) and Parkin were detected using anti-SUMO, anti-FLAG and anti-Parkin antibodies respectively. ( C ) Activation of ΔUbl-Parkin by TcPINK1 is abolished by S65A ubiquitin. ΔUbl-Parkin was incubated in the presence or absence of wild-type (WT) or kinase-inactive (KI) PINK1. The ubiquitylation reactions were then initiated by the addition of ubiquitylation assay components including 0.04 mM wild-type (WT) or S65A His 6 –FLAG–ubiquitin. Reactions were terminated after 60 min by the addition of SDS loading buffer and products were analysed by SDS/PAGE. Miro1, ubiquitin and Parkin were detected using anti-SUMO, anti-FLAG and anti-Parkin antibodies respectively. The molecular mass in kDa is indicated.

    Journal: Biochemical Journal

    Article Title: Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65

    doi: 10.1042/BJ20140334

    Figure Lengend Snippet: PINK1-dependent phosphorylation of ubiquitin at Ser 65 leads to increased activity of ΔUbl-Parkin ( A ) MBP–TcPINK1 enhances ΔUbl-Parkin-mediated ubiquitin discharge from UbcH7. ΔUbl-Parkin was phosphorylated using wild-type (WT), kinase-inactive (KI) or no MBP–TcPINK1 in the presence of Mg 2+ -[γ- 32 P]ATP. An E2-discharge assay was established by incubation of this mixture with 2 μg of UbcH7 that had been pre-incubated with E1 and FLAG–ubiquitin in the presence of ATP for 60 min. Reactions were allowed to continue for 15 min and were stopped using SDS loading buffer in the absence of reducing agent. Samples were analysed as described in the Materials and methods section. Protein phosphorylation was monitored by autoradiography (bottom panel). ( B ) ΔUbl-Parkin activity is increased by wild-type TcPINK1. A 2 μg amount of full-length (WT) or ΔUbl-Parkin was incubated with 1 μg of wild-type (WT), kinase-inactive (KI) or no TcPINK1 in a kinase reaction for 60 min. The ubiquitylation reactions were then initiated by the addition of ubiquitylation assay components (E1, UbcH7 and FLAG–ubiquitin) and 2 μg of His 6 –SUMO-Miro1. Reactions were terminated after 60 min by the addition of SDS loading buffer and products were analysed by SDS/PAGE. Miro1, ubiquitin (Ub) and Parkin were detected using anti-SUMO, anti-FLAG and anti-Parkin antibodies respectively. ( C ) Activation of ΔUbl-Parkin by TcPINK1 is abolished by S65A ubiquitin. ΔUbl-Parkin was incubated in the presence or absence of wild-type (WT) or kinase-inactive (KI) PINK1. The ubiquitylation reactions were then initiated by the addition of ubiquitylation assay components including 0.04 mM wild-type (WT) or S65A His 6 –FLAG–ubiquitin. Reactions were terminated after 60 min by the addition of SDS loading buffer and products were analysed by SDS/PAGE. Miro1, ubiquitin and Parkin were detected using anti-SUMO, anti-FLAG and anti-Parkin antibodies respectively. The molecular mass in kDa is indicated.

    Article Snippet: Incorporation of [γ-32 P]ATP into substrates was analysed by autoradiography using Amersham Hyperfilm.

    Techniques: Activity Assay, Incubation, Autoradiography, Ubiquitin Assay, SDS Page, Activation Assay