ratp Promega Search Results


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  • 99
    Promega ratp
    Ratp, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 438 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Promega ratp standard
    Ratp Standard, supplied by Promega, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Promega rntp
    Rntp, supplied by Promega, used in various techniques. Bioz Stars score: 84/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Promega ligase mastermix
    Ligase Mastermix, supplied by Promega, used in various techniques. Bioz Stars score: 81/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Promega ribonucleotide triphosphates
    Ribonucleotide Triphosphates, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Promega kinase glo kit
    Kinase Glo Kit, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Promega kinase glo luciferase kit
    Kinase Glo Luciferase Kit, supplied by Promega, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega magnesium acetate
    Magnesium Acetate, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 358 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Promega kinase glo luciferase assay
    Kinase Glo Luciferase Assay, supplied by Promega, used in various techniques. Bioz Stars score: 80/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Promega gsk 3β assay kinase glo kit
    Linear graph of IC 50 assay of <t>GSK-3β</t> treated with selected Amaryllidaceae alkaloids. Concentrations of alkaloids were 6.25; 12.5; 25; 50 and 100 μM. Activity is presented as % inhibition.
    Gsk 3β Assay Kinase Glo Kit, supplied by Promega, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Promega recombinant gst gck
    Parallel assay development strategy to interrogate recombinant <t>GCK</t> and GKRP. Human GCK and GKRP were affinity-purified using <t>GST</t> and FLAG tags, respectively. (A) FRET-based (HTRF) detection. Antibodies recognizing the affinity tags are conjugated to FRET donor and acceptor molecules. Excitation of the donor results in energy transfer (FRET; red dashed oval) to the acceptor only if the acceptor and donor are in close proximity. (B) Reaction scheme for G6PDH/diaphorase dual-coupled assay. The generation of the fluorescent product resorufin (red dashed oval) is measured as the reaction progresses in real time by excitation at 525 nm with emission at 590 nm. (C) Reaction scheme for coupling of ADP generation by GCK to luminescence-based detection. The GCK reaction is allowed to run for a set period of time, and the reaction is then terminated and a two-step reaction utilizes Ultra-Glo™ firefly luciferase to generate bioluminescence (red dashed oval). Reagent 1: ADP-Glo™ Reagent; Reagent 2: Kinase Detection Reagent.
    Recombinant Gst Gck, supplied by Promega, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Promega kinase glo plus luminescent assay kit
    Parallel assay development strategy to interrogate recombinant <t>GCK</t> and GKRP. Human GCK and GKRP were affinity-purified using <t>GST</t> and FLAG tags, respectively. (A) FRET-based (HTRF) detection. Antibodies recognizing the affinity tags are conjugated to FRET donor and acceptor molecules. Excitation of the donor results in energy transfer (FRET; red dashed oval) to the acceptor only if the acceptor and donor are in close proximity. (B) Reaction scheme for G6PDH/diaphorase dual-coupled assay. The generation of the fluorescent product resorufin (red dashed oval) is measured as the reaction progresses in real time by excitation at 525 nm with emission at 590 nm. (C) Reaction scheme for coupling of ADP generation by GCK to luminescence-based detection. The GCK reaction is allowed to run for a set period of time, and the reaction is then terminated and a two-step reaction utilizes Ultra-Glo™ firefly luciferase to generate bioluminescence (red dashed oval). Reagent 1: ADP-Glo™ Reagent; Reagent 2: Kinase Detection Reagent.
    Kinase Glo Plus Luminescent Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 82/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega kinase glo luminescent assay regents
    Parallel assay development strategy to interrogate recombinant <t>GCK</t> and GKRP. Human GCK and GKRP were affinity-purified using <t>GST</t> and FLAG tags, respectively. (A) FRET-based (HTRF) detection. Antibodies recognizing the affinity tags are conjugated to FRET donor and acceptor molecules. Excitation of the donor results in energy transfer (FRET; red dashed oval) to the acceptor only if the acceptor and donor are in close proximity. (B) Reaction scheme for G6PDH/diaphorase dual-coupled assay. The generation of the fluorescent product resorufin (red dashed oval) is measured as the reaction progresses in real time by excitation at 525 nm with emission at 590 nm. (C) Reaction scheme for coupling of ADP generation by GCK to luminescence-based detection. The GCK reaction is allowed to run for a set period of time, and the reaction is then terminated and a two-step reaction utilizes Ultra-Glo™ firefly luciferase to generate bioluminescence (red dashed oval). Reagent 1: ADP-Glo™ Reagent; Reagent 2: Kinase Detection Reagent.
    Kinase Glo Luminescent Assay Regents, supplied by Promega, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega adp glo max assay
    Parallel assay development strategy to interrogate recombinant <t>GCK</t> and GKRP. Human GCK and GKRP were affinity-purified using <t>GST</t> and FLAG tags, respectively. (A) FRET-based (HTRF) detection. Antibodies recognizing the affinity tags are conjugated to FRET donor and acceptor molecules. Excitation of the donor results in energy transfer (FRET; red dashed oval) to the acceptor only if the acceptor and donor are in close proximity. (B) Reaction scheme for G6PDH/diaphorase dual-coupled assay. The generation of the fluorescent product resorufin (red dashed oval) is measured as the reaction progresses in real time by excitation at 525 nm with emission at 590 nm. (C) Reaction scheme for coupling of ADP generation by GCK to luminescence-based detection. The GCK reaction is allowed to run for a set period of time, and the reaction is then terminated and a two-step reaction utilizes Ultra-Glo™ firefly luciferase to generate bioluminescence (red dashed oval). Reagent 1: ADP-Glo™ Reagent; Reagent 2: Kinase Detection Reagent.
    Adp Glo Max Assay, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Promega luciferase atp consumption assay
    Binding modes of ligands bound to Pf KRS1 and Cp <t>KRS.</t> ( A ) Pf KRS1:Lys: 2 showing the binding mode of 2 (C atoms, gold) bound to the <t>ATP</t> site of Pf KRS1 (PDB ID code 6AGT) superimposed upon Pf KRS1:Lys:cladosporin (cladosporin C atoms, slate; PDB ID code 4PG3). ( B ) Pf KRS1: 5 showing binding mode of 5 bound to Pf KRS1 (PDB ID code 6HCU). ( C ) Overlay of Cp KRS:Lys:cladosporin (C atoms, gold; PDB ID code 5ELO) compared with Pf KRS1:Lys:cladosporin (C atoms, gray; PDB ID code 4PG3). Nonconserved residues within the ligand binding site are labeled. ( D ) Cp KRS:Lys: 5 showing binding mode of 5 (C atoms, gold) in complex with Cp KRS:Lys (C atoms, gray; PDB ID code 6HCW). H-bonds are shown as dashed lines, and key residues are labeled for clarity.
    Luciferase Atp Consumption Assay, supplied by Promega, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    70
    Promega adp glo in vitro kinase assay
    PfCK2 phosphorylates PfHP1 in vitro . ( a ) Coomassie-stained SDS-PAGE gel showing the purified recombinant PfHP1 and PfCD.H proteins. ( b ) In vitro <t>ADP-Glo</t> assay results reveal that PfCK2 phosphorylates PfHP1 and PfCD.H in vitro . The percentage of ADP converted back into ATP (y-axis) is a surrogate measure for kinase activity (i.e. the relative amount of ATP consumed in the kinase reaction). β-casein was included as a positive control substrate for PfCK2. Recombinant PfHP1 and PfCD.H in absence of PfCK2 were used as negative controls. Values represent the average of two replicate reactions. Error bars represent SD. ( c ) Cropped sections of a Coomassie-stained SDS-PAGE gel (top) and the corresponding autoradiogram (bottom) of the in vitro γ-P 32 -ATP PfCK2 kinase assay performed with recombinant PfHP1 and PfCD.H substrates. β-casein was used as a positive control substrate. 20 µM TBB was used as a specific inhibitor of PfCK2 50 , 56 . The full-size Coomassie-stained gel and autoradiogram are shown in Supplementary Fig. 6 .
    Adp Glo In Vitro Kinase Assay, supplied by Promega, used in various techniques. Bioz Stars score: 70/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    70
    Promega kinase glo r luminescent kinase assay kit
    PfCK2 phosphorylates PfHP1 in vitro . ( a ) Coomassie-stained SDS-PAGE gel showing the purified recombinant PfHP1 and PfCD.H proteins. ( b ) In vitro <t>ADP-Glo</t> assay results reveal that PfCK2 phosphorylates PfHP1 and PfCD.H in vitro . The percentage of ADP converted back into ATP (y-axis) is a surrogate measure for kinase activity (i.e. the relative amount of ATP consumed in the kinase reaction). β-casein was included as a positive control substrate for PfCK2. Recombinant PfHP1 and PfCD.H in absence of PfCK2 were used as negative controls. Values represent the average of two replicate reactions. Error bars represent SD. ( c ) Cropped sections of a Coomassie-stained SDS-PAGE gel (top) and the corresponding autoradiogram (bottom) of the in vitro γ-P 32 -ATP PfCK2 kinase assay performed with recombinant PfHP1 and PfCD.H substrates. β-casein was used as a positive control substrate. 20 µM TBB was used as a specific inhibitor of PfCK2 50 , 56 . The full-size Coomassie-stained gel and autoradiogram are shown in Supplementary Fig. 6 .
    Kinase Glo R Luminescent Kinase Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 70/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Promega olomoucine
    CDKN3 modulates progression through mitosis via regulation of CDC2 phosphorylation at Thr-161. (A) The CDKN3 interface is conserved in human CDK2 (Protein Database [PDB] accession no. 1B39 ) and CDC2 (PDB accession no. 3LFQ ) kinases (GDSEID/DYK motifs, blue). Activation loops (magenta) include putative CDKN3 target residue (pThr160/161; yellow). CDC25 target sites are green. Regions of full conservation are red; other regions are gray. (B) Endogenous CDKN3 colocalizes with endogenous CDC2 on centrosomes during mitosis. (C) Endogenous CDC2 pThr161 localizes to centrosomes and the mitotic spindle during cell division. Dephosphorylation of CDC2 pThr161 occurs in anaphase. HeLa cells were stained with antibody recognizing CDC2 pThr-161 , anti–α-tubulin antibody, and Hoechst 33342. (D) CDC pThr161 localizes to kinetochores in early mitosis. CDC2 pThr161 (red) colocalizes with the kinetochore marker GFP-CENPA (green) in prometaphase but not in anaphase. (E) CDC2 pThr161 is dephosphorylated at exit from mitosis. Cells were arrested in G2 through 24 h of exposure to RO3306 and washed to trigger mitotic entry. Decreasing cyclin B1 levels indicate cell cycle progression toward the mitotic exit. (F) Hyperphosphorylation of CDC2 pThr161 in HeLa cells transfected with CDKN3 siRNA. Total CDC2 and CDC2 pTyr15 levels are unaffected by CDKN3 siRNA. (G) Recombinant CDKN3 inactivates recombinant CDC2/cyclin B in an in vitro kinase assay in a dose-dependent manner. Increasing amounts of recombinant CDKN3 (0.5–5 µg) were incubated for 30 min in kinase buffer with a constant amount of active CDC2–cyclin B kinase complex, CDC2 substrate (histone H1), and radioactive [P 32 ]γ-ATP. CDC2-dependent H1 phosphorylation was detected by autoradiography. 150 mM <t>olomoucine</t> (a CDK kinase inhibitor) was used as a control. (H) Cells transfected with CDKN3 siRNA fail to dephosphorylate CDC2 pThr161 in early anaphase. Note the normal CDC2 pThr-161 metaphase signal in control and CDKN3 siRNA cells. The CDC2 pThr-161 signal persists in CDKN3 siRNA cells during anaphase.
    Olomoucine, supplied by Promega, used in various techniques. Bioz Stars score: 82/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Promega adp glo melk kinase assay kit
    Catalytic activity of <t>MELK</t> is required for HIV-1 infection. (A) Effect of exogenous wt or T167A MELK on single-round HIV-1 infection. VSV-G-pseudotyped NL4-3luc was used to infect parental MT4C5 (white bar: lane 1), Non-T (gray bars: lanes 2 and 9) and MELK-KD-1 (dark gray bars: lanes 3 to 8) cells transduced with control vector (lane 4), wild - type MELK (lanes 5 and 6) or catalytically inactive T167A MELK mutant (lanes 7 and 8) (see also S7 Fig ). Two independent MELK-KD-1 cell pools expressing wild - type MELK (lanes 5 and 6) or T167A MELK mutant (lanes 7 and 8) were used. Error bars indicate the standard deviations calculated from five independent experiments. (B) In vitro luminescent kinase assay with recombinant active MELK (10 or 100 ng) and increasing amounts of ZIPtide, a substrate for MELK (upper panel). Phosphorylation of the substrate was monitored as the amount of <t>ADP</t> produced during the kinase reaction. Effect of OTSSP167, a MELK kinase inhibitor, on in vitro MELK kinase activity (lower panel). Error bars indicate the standard deviations calculated from three independent experiments. (C) In vitro luminescent kinase assay with recombinant active MELK and increasing amounts of the indicated GST fusion proteins in the presence or absence of OTSSP167 (100 nM). Mean values from five independent experiments are shown. Error bars indicate the standard deviations calculated from five independent experiments. (D) In vitro luminescent kinase assay with recombinant active MELK and increasing amounts of the indicated substrates in the presence or absence of OTSSP167 (100 nM). Phosphorylation of proteins was monitored as in (C) . Error bars indicate the standard deviations calculated from five independent experiments. (E) List of fifteen different peptides containing serine or threonine residues in HIV-1 CA. (F) In vitro luminescent kinase assay with recombinant active MELK and increasing amounts of each peptide shown in (E) . Phosphorylation of the peptides was monitored as in (B) . Experiments were performed at least three times and error bars are standard deviations calculated from three independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test (A) , or two-way ANOVA with Tukey’s multiple comparison test (C and D) . ns, not significant ( P > 0.05); * P
    Adp Glo Melk Kinase Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Linear graph of IC 50 assay of GSK-3β treated with selected Amaryllidaceae alkaloids. Concentrations of alkaloids were 6.25; 12.5; 25; 50 and 100 μM. Activity is presented as % inhibition.

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Amaryllidaceae Alkaloids as Potential Glycogen Synthase Kinase-3β Inhibitors

    doi: 10.3390/molecules23040719

    Figure Lengend Snippet: Linear graph of IC 50 assay of GSK-3β treated with selected Amaryllidaceae alkaloids. Concentrations of alkaloids were 6.25; 12.5; 25; 50 and 100 μM. Activity is presented as % inhibition.

    Article Snippet: GSK-3β Assay Kinase-Glo Kit was obtained from Promega (Promega Biotech Iberica, S.L., Madrid, Spain), and human recombinant GSK-3β and GSM substrate mimicking Glycogen Muscle Synthase from Merck Millipore (Darmstadt, Germany).

    Techniques: Activity Assay, Inhibition

    Parallel assay development strategy to interrogate recombinant GCK and GKRP. Human GCK and GKRP were affinity-purified using GST and FLAG tags, respectively. (A) FRET-based (HTRF) detection. Antibodies recognizing the affinity tags are conjugated to FRET donor and acceptor molecules. Excitation of the donor results in energy transfer (FRET; red dashed oval) to the acceptor only if the acceptor and donor are in close proximity. (B) Reaction scheme for G6PDH/diaphorase dual-coupled assay. The generation of the fluorescent product resorufin (red dashed oval) is measured as the reaction progresses in real time by excitation at 525 nm with emission at 590 nm. (C) Reaction scheme for coupling of ADP generation by GCK to luminescence-based detection. The GCK reaction is allowed to run for a set period of time, and the reaction is then terminated and a two-step reaction utilizes Ultra-Glo™ firefly luciferase to generate bioluminescence (red dashed oval). Reagent 1: ADP-Glo™ Reagent; Reagent 2: Kinase Detection Reagent.

    Journal: PLoS ONE

    Article Title: A Panel of Diverse Assays to Interrogate the Interaction between Glucokinase and Glucokinase Regulatory Protein, Two Vital Proteins in Human Disease

    doi: 10.1371/journal.pone.0089335

    Figure Lengend Snippet: Parallel assay development strategy to interrogate recombinant GCK and GKRP. Human GCK and GKRP were affinity-purified using GST and FLAG tags, respectively. (A) FRET-based (HTRF) detection. Antibodies recognizing the affinity tags are conjugated to FRET donor and acceptor molecules. Excitation of the donor results in energy transfer (FRET; red dashed oval) to the acceptor only if the acceptor and donor are in close proximity. (B) Reaction scheme for G6PDH/diaphorase dual-coupled assay. The generation of the fluorescent product resorufin (red dashed oval) is measured as the reaction progresses in real time by excitation at 525 nm with emission at 590 nm. (C) Reaction scheme for coupling of ADP generation by GCK to luminescence-based detection. The GCK reaction is allowed to run for a set period of time, and the reaction is then terminated and a two-step reaction utilizes Ultra-Glo™ firefly luciferase to generate bioluminescence (red dashed oval). Reagent 1: ADP-Glo™ Reagent; Reagent 2: Kinase Detection Reagent.

    Article Snippet: Bioluminescence Assays A luminescence-based assay was developed for detection of ADP generation by recombinant GST-GCK (ADP-Glo™ Kinase Assay; Promega).

    Techniques: Recombinant, Affinity Purification, Luciferase

    Binding modes of ligands bound to Pf KRS1 and Cp KRS. ( A ) Pf KRS1:Lys: 2 showing the binding mode of 2 (C atoms, gold) bound to the ATP site of Pf KRS1 (PDB ID code 6AGT) superimposed upon Pf KRS1:Lys:cladosporin (cladosporin C atoms, slate; PDB ID code 4PG3). ( B ) Pf KRS1: 5 showing binding mode of 5 bound to Pf KRS1 (PDB ID code 6HCU). ( C ) Overlay of Cp KRS:Lys:cladosporin (C atoms, gold; PDB ID code 5ELO) compared with Pf KRS1:Lys:cladosporin (C atoms, gray; PDB ID code 4PG3). Nonconserved residues within the ligand binding site are labeled. ( D ) Cp KRS:Lys: 5 showing binding mode of 5 (C atoms, gold) in complex with Cp KRS:Lys (C atoms, gray; PDB ID code 6HCW). H-bonds are shown as dashed lines, and key residues are labeled for clarity.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis

    doi: 10.1073/pnas.1814685116

    Figure Lengend Snippet: Binding modes of ligands bound to Pf KRS1 and Cp KRS. ( A ) Pf KRS1:Lys: 2 showing the binding mode of 2 (C atoms, gold) bound to the ATP site of Pf KRS1 (PDB ID code 6AGT) superimposed upon Pf KRS1:Lys:cladosporin (cladosporin C atoms, slate; PDB ID code 4PG3). ( B ) Pf KRS1: 5 showing binding mode of 5 bound to Pf KRS1 (PDB ID code 6HCU). ( C ) Overlay of Cp KRS:Lys:cladosporin (C atoms, gold; PDB ID code 5ELO) compared with Pf KRS1:Lys:cladosporin (C atoms, gray; PDB ID code 4PG3). Nonconserved residues within the ligand binding site are labeled. ( D ) Cp KRS:Lys: 5 showing binding mode of 5 (C atoms, gold) in complex with Cp KRS:Lys (C atoms, gray; PDB ID code 6HCW). H-bonds are shown as dashed lines, and key residues are labeled for clarity.

    Article Snippet: We produced recombinant Pf KRS1 (77–583 and 80–583), Cp KRS (46-end), and Hs KRS (full-length) proteins and developed biochemical assays based on the luciferase ATP consumption assay (Kinase-Glo; Promega) , which was suitable for the high-throughput screening, and the pyrophosphate generation assay (EnzChek) ( ) format for kinetic characterization of the enzymes.

    Techniques: Binding Assay, Ligand Binding Assay, Labeling

    PfCK2 phosphorylates PfHP1 in vitro . ( a ) Coomassie-stained SDS-PAGE gel showing the purified recombinant PfHP1 and PfCD.H proteins. ( b ) In vitro ADP-Glo assay results reveal that PfCK2 phosphorylates PfHP1 and PfCD.H in vitro . The percentage of ADP converted back into ATP (y-axis) is a surrogate measure for kinase activity (i.e. the relative amount of ATP consumed in the kinase reaction). β-casein was included as a positive control substrate for PfCK2. Recombinant PfHP1 and PfCD.H in absence of PfCK2 were used as negative controls. Values represent the average of two replicate reactions. Error bars represent SD. ( c ) Cropped sections of a Coomassie-stained SDS-PAGE gel (top) and the corresponding autoradiogram (bottom) of the in vitro γ-P 32 -ATP PfCK2 kinase assay performed with recombinant PfHP1 and PfCD.H substrates. β-casein was used as a positive control substrate. 20 µM TBB was used as a specific inhibitor of PfCK2 50 , 56 . The full-size Coomassie-stained gel and autoradiogram are shown in Supplementary Fig. 6 .

    Journal: Scientific Reports

    Article Title: Mapping and functional analysis of heterochromatin protein 1 phosphorylation in the malaria parasite Plasmodium falciparum

    doi: 10.1038/s41598-019-53325-9

    Figure Lengend Snippet: PfCK2 phosphorylates PfHP1 in vitro . ( a ) Coomassie-stained SDS-PAGE gel showing the purified recombinant PfHP1 and PfCD.H proteins. ( b ) In vitro ADP-Glo assay results reveal that PfCK2 phosphorylates PfHP1 and PfCD.H in vitro . The percentage of ADP converted back into ATP (y-axis) is a surrogate measure for kinase activity (i.e. the relative amount of ATP consumed in the kinase reaction). β-casein was included as a positive control substrate for PfCK2. Recombinant PfHP1 and PfCD.H in absence of PfCK2 were used as negative controls. Values represent the average of two replicate reactions. Error bars represent SD. ( c ) Cropped sections of a Coomassie-stained SDS-PAGE gel (top) and the corresponding autoradiogram (bottom) of the in vitro γ-P 32 -ATP PfCK2 kinase assay performed with recombinant PfHP1 and PfCD.H substrates. β-casein was used as a positive control substrate. 20 µM TBB was used as a specific inhibitor of PfCK2 50 , 56 . The full-size Coomassie-stained gel and autoradiogram are shown in Supplementary Fig. 6 .

    Article Snippet: The ADP-Glo in vitro kinase assay was performed in duplicates according to the manufacturer’s instructions (Promega, USA).

    Techniques: In Vitro, Staining, SDS Page, Purification, Recombinant, Glo Assay, Activity Assay, Positive Control, Kinase Assay

    CDKN3 modulates progression through mitosis via regulation of CDC2 phosphorylation at Thr-161. (A) The CDKN3 interface is conserved in human CDK2 (Protein Database [PDB] accession no. 1B39 ) and CDC2 (PDB accession no. 3LFQ ) kinases (GDSEID/DYK motifs, blue). Activation loops (magenta) include putative CDKN3 target residue (pThr160/161; yellow). CDC25 target sites are green. Regions of full conservation are red; other regions are gray. (B) Endogenous CDKN3 colocalizes with endogenous CDC2 on centrosomes during mitosis. (C) Endogenous CDC2 pThr161 localizes to centrosomes and the mitotic spindle during cell division. Dephosphorylation of CDC2 pThr161 occurs in anaphase. HeLa cells were stained with antibody recognizing CDC2 pThr-161 , anti–α-tubulin antibody, and Hoechst 33342. (D) CDC pThr161 localizes to kinetochores in early mitosis. CDC2 pThr161 (red) colocalizes with the kinetochore marker GFP-CENPA (green) in prometaphase but not in anaphase. (E) CDC2 pThr161 is dephosphorylated at exit from mitosis. Cells were arrested in G2 through 24 h of exposure to RO3306 and washed to trigger mitotic entry. Decreasing cyclin B1 levels indicate cell cycle progression toward the mitotic exit. (F) Hyperphosphorylation of CDC2 pThr161 in HeLa cells transfected with CDKN3 siRNA. Total CDC2 and CDC2 pTyr15 levels are unaffected by CDKN3 siRNA. (G) Recombinant CDKN3 inactivates recombinant CDC2/cyclin B in an in vitro kinase assay in a dose-dependent manner. Increasing amounts of recombinant CDKN3 (0.5–5 µg) were incubated for 30 min in kinase buffer with a constant amount of active CDC2–cyclin B kinase complex, CDC2 substrate (histone H1), and radioactive [P 32 ]γ-ATP. CDC2-dependent H1 phosphorylation was detected by autoradiography. 150 mM olomoucine (a CDK kinase inhibitor) was used as a control. (H) Cells transfected with CDKN3 siRNA fail to dephosphorylate CDC2 pThr161 in early anaphase. Note the normal CDC2 pThr-161 metaphase signal in control and CDKN3 siRNA cells. The CDC2 pThr-161 signal persists in CDKN3 siRNA cells during anaphase.

    Journal: The Journal of Cell Biology

    Article Title: The tumor suppressor CDKN3 controls mitosis

    doi: 10.1083/jcb.201205125

    Figure Lengend Snippet: CDKN3 modulates progression through mitosis via regulation of CDC2 phosphorylation at Thr-161. (A) The CDKN3 interface is conserved in human CDK2 (Protein Database [PDB] accession no. 1B39 ) and CDC2 (PDB accession no. 3LFQ ) kinases (GDSEID/DYK motifs, blue). Activation loops (magenta) include putative CDKN3 target residue (pThr160/161; yellow). CDC25 target sites are green. Regions of full conservation are red; other regions are gray. (B) Endogenous CDKN3 colocalizes with endogenous CDC2 on centrosomes during mitosis. (C) Endogenous CDC2 pThr161 localizes to centrosomes and the mitotic spindle during cell division. Dephosphorylation of CDC2 pThr161 occurs in anaphase. HeLa cells were stained with antibody recognizing CDC2 pThr-161 , anti–α-tubulin antibody, and Hoechst 33342. (D) CDC pThr161 localizes to kinetochores in early mitosis. CDC2 pThr161 (red) colocalizes with the kinetochore marker GFP-CENPA (green) in prometaphase but not in anaphase. (E) CDC2 pThr161 is dephosphorylated at exit from mitosis. Cells were arrested in G2 through 24 h of exposure to RO3306 and washed to trigger mitotic entry. Decreasing cyclin B1 levels indicate cell cycle progression toward the mitotic exit. (F) Hyperphosphorylation of CDC2 pThr161 in HeLa cells transfected with CDKN3 siRNA. Total CDC2 and CDC2 pTyr15 levels are unaffected by CDKN3 siRNA. (G) Recombinant CDKN3 inactivates recombinant CDC2/cyclin B in an in vitro kinase assay in a dose-dependent manner. Increasing amounts of recombinant CDKN3 (0.5–5 µg) were incubated for 30 min in kinase buffer with a constant amount of active CDC2–cyclin B kinase complex, CDC2 substrate (histone H1), and radioactive [P 32 ]γ-ATP. CDC2-dependent H1 phosphorylation was detected by autoradiography. 150 mM olomoucine (a CDK kinase inhibitor) was used as a control. (H) Cells transfected with CDKN3 siRNA fail to dephosphorylate CDC2 pThr161 in early anaphase. Note the normal CDC2 pThr-161 metaphase signal in control and CDKN3 siRNA cells. The CDC2 pThr-161 signal persists in CDKN3 siRNA cells during anaphase.

    Article Snippet: Olomoucine (a small-molecule CDK kinase inhibitor used as a negative control) was purchased from Promega.

    Techniques: Activation Assay, De-Phosphorylation Assay, Staining, Marker, Transfection, Recombinant, In Vitro, Kinase Assay, Incubation, Autoradiography

    Catalytic activity of MELK is required for HIV-1 infection. (A) Effect of exogenous wt or T167A MELK on single-round HIV-1 infection. VSV-G-pseudotyped NL4-3luc was used to infect parental MT4C5 (white bar: lane 1), Non-T (gray bars: lanes 2 and 9) and MELK-KD-1 (dark gray bars: lanes 3 to 8) cells transduced with control vector (lane 4), wild - type MELK (lanes 5 and 6) or catalytically inactive T167A MELK mutant (lanes 7 and 8) (see also S7 Fig ). Two independent MELK-KD-1 cell pools expressing wild - type MELK (lanes 5 and 6) or T167A MELK mutant (lanes 7 and 8) were used. Error bars indicate the standard deviations calculated from five independent experiments. (B) In vitro luminescent kinase assay with recombinant active MELK (10 or 100 ng) and increasing amounts of ZIPtide, a substrate for MELK (upper panel). Phosphorylation of the substrate was monitored as the amount of ADP produced during the kinase reaction. Effect of OTSSP167, a MELK kinase inhibitor, on in vitro MELK kinase activity (lower panel). Error bars indicate the standard deviations calculated from three independent experiments. (C) In vitro luminescent kinase assay with recombinant active MELK and increasing amounts of the indicated GST fusion proteins in the presence or absence of OTSSP167 (100 nM). Mean values from five independent experiments are shown. Error bars indicate the standard deviations calculated from five independent experiments. (D) In vitro luminescent kinase assay with recombinant active MELK and increasing amounts of the indicated substrates in the presence or absence of OTSSP167 (100 nM). Phosphorylation of proteins was monitored as in (C) . Error bars indicate the standard deviations calculated from five independent experiments. (E) List of fifteen different peptides containing serine or threonine residues in HIV-1 CA. (F) In vitro luminescent kinase assay with recombinant active MELK and increasing amounts of each peptide shown in (E) . Phosphorylation of the peptides was monitored as in (B) . Experiments were performed at least three times and error bars are standard deviations calculated from three independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test (A) , or two-way ANOVA with Tukey’s multiple comparison test (C and D) . ns, not significant ( P > 0.05); * P

    Journal: PLoS Pathogens

    Article Title: Phosphorylation of the HIV-1 capsid by MELK triggers uncoating to promote viral cDNA synthesis

    doi: 10.1371/journal.ppat.1006441

    Figure Lengend Snippet: Catalytic activity of MELK is required for HIV-1 infection. (A) Effect of exogenous wt or T167A MELK on single-round HIV-1 infection. VSV-G-pseudotyped NL4-3luc was used to infect parental MT4C5 (white bar: lane 1), Non-T (gray bars: lanes 2 and 9) and MELK-KD-1 (dark gray bars: lanes 3 to 8) cells transduced with control vector (lane 4), wild - type MELK (lanes 5 and 6) or catalytically inactive T167A MELK mutant (lanes 7 and 8) (see also S7 Fig ). Two independent MELK-KD-1 cell pools expressing wild - type MELK (lanes 5 and 6) or T167A MELK mutant (lanes 7 and 8) were used. Error bars indicate the standard deviations calculated from five independent experiments. (B) In vitro luminescent kinase assay with recombinant active MELK (10 or 100 ng) and increasing amounts of ZIPtide, a substrate for MELK (upper panel). Phosphorylation of the substrate was monitored as the amount of ADP produced during the kinase reaction. Effect of OTSSP167, a MELK kinase inhibitor, on in vitro MELK kinase activity (lower panel). Error bars indicate the standard deviations calculated from three independent experiments. (C) In vitro luminescent kinase assay with recombinant active MELK and increasing amounts of the indicated GST fusion proteins in the presence or absence of OTSSP167 (100 nM). Mean values from five independent experiments are shown. Error bars indicate the standard deviations calculated from five independent experiments. (D) In vitro luminescent kinase assay with recombinant active MELK and increasing amounts of the indicated substrates in the presence or absence of OTSSP167 (100 nM). Phosphorylation of proteins was monitored as in (C) . Error bars indicate the standard deviations calculated from five independent experiments. (E) List of fifteen different peptides containing serine or threonine residues in HIV-1 CA. (F) In vitro luminescent kinase assay with recombinant active MELK and increasing amounts of each peptide shown in (E) . Phosphorylation of the peptides was monitored as in (B) . Experiments were performed at least three times and error bars are standard deviations calculated from three independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test (A) , or two-way ANOVA with Tukey’s multiple comparison test (C and D) . ns, not significant ( P > 0.05); * P

    Article Snippet: In vitro phosphorylation assay In vitro phosphorylation assays were performed with the ADP-Glo MELK kinase assay kit, following the manufacturer’s instructions (Promega Corp, Madison, WI).

    Techniques: Activity Assay, Infection, Transduction, Plasmid Preparation, Mutagenesis, Expressing, In Vitro, Kinase Assay, Recombinant, Produced