ppase  (New England Biolabs)


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    Name:
    Pyrophosphatase inorganic yeast
    Description:
    Pyrophosphatase inorganic yeast 50 units
    Catalog Number:
    m2403l
    Price:
    273
    Size:
    50 units
    Category:
    Other Enzymes
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    Structured Review

    New England Biolabs ppase
    Pyrophosphatase inorganic yeast
    Pyrophosphatase inorganic yeast 50 units
    https://www.bioz.com/result/ppase/product/New England Biolabs
    Average 98 stars, based on 50 article reviews
    Price from $9.99 to $1999.99
    ppase - by Bioz Stars, 2020-10
    98/100 stars

    Images

    1) Product Images from "M-Phase Phosphoprotein 9 regulates ciliogenesis by modulating CP110-CEP97 complex localization at the mother centriole"

    Article Title: M-Phase Phosphoprotein 9 regulates ciliogenesis by modulating CP110-CEP97 complex localization at the mother centriole

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06990-9

    TTBK2 phosphorylates MPP9 mainly at S629 site. a Immunoblots of lysates from HEK293T cells co-overexpressing Flag-MPP9 and the indicated cilia-related kinase. Tubulin was used as a loading control. b Immunoblots of lysates from HEK293T cells co-overexpressing Flag-tagged MPP9 full-length (FL) or the indicated MPP9 truncation mutants with TTBK2-GFP-WT or TTBK2-GFP-KD. Tubulin was used as a loading control. WT, wild-type; KD, kinase-dead. c Immunoblots of lysates from HEK293T cells co-overexpressing Flag-tagged MPP9 (401–800 aa) and TTBK2-GFP-WT or TTBK2-GFP-KD after treatment with λ-PPase. Tubulin was used as a loading control. d Mass spectrometric analysis showing phosphorylation sites of MPP9 at S629 and S636. e Immunoblots of lysates from HEK293T cells co-overexpressing TTBK2-GFP and Flag-MPP9 (401–800 aa)-WT, or the indicated unphosphorylatable mutants. Tubulin was used as a loading control. f GST-MPP9 (401–800 aa)-WT, or the indicated mutants were subjected to a TTBK2 kinase assay in vitro followed by autoradiography. Coomassie blue (CBB) staining showed GST-tagged proteins. Relative amounts of phosphorylated MPP9 signals were quantified. g Lysates of HEK293T cells co-overexpressing Flag-tagged MPP9-WT or the S629A mutant and TTBK2-GFP were subjected to immunoprecipitation (IP) and immunoblotting with anti-Flag and anti-Phospho-S629 antibodies. Relative amounts of S629-phosphorylated MPP9 were quantified. h Immunostaining of S629-phosphorylated MPP9 (red) and Flag in Flag-MPP9 (green, upper) or Flag-CEP170 (green, lower) overexpressing hTERT RPE-1 cells. i Immunostaining of S629-phosphorylated MPP9 (red) and Flag in Flag-MPP9 (green) or Flag-MPP9–629A (green) overexpressing hTERT RPE-1 cells after depleting MPP9. j Quantifications of S629-phosphorylated MPP9 signals in i ( n = 50 cells for each group). k Immunostaining of S629-phosphorylated MPP9 (green) and Centrin-3 (red) in control- or TTBK2-siRNA transfected hTERT RPE-1 cells. l Quantifications of S629-phosphorylated MPP9 signals in k ( n = 50 cells for each group). m Lysates of control-siRNA or TTBK2-siRNA transfected hTERT RPE-1 cells after serum starvation were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. Relative amounts of S629-phosphorylated MPP9 were quantified and normalized to MPP9. For j , l , bars represent the means ± S.E.M for three independent experiments. *** p
    Figure Legend Snippet: TTBK2 phosphorylates MPP9 mainly at S629 site. a Immunoblots of lysates from HEK293T cells co-overexpressing Flag-MPP9 and the indicated cilia-related kinase. Tubulin was used as a loading control. b Immunoblots of lysates from HEK293T cells co-overexpressing Flag-tagged MPP9 full-length (FL) or the indicated MPP9 truncation mutants with TTBK2-GFP-WT or TTBK2-GFP-KD. Tubulin was used as a loading control. WT, wild-type; KD, kinase-dead. c Immunoblots of lysates from HEK293T cells co-overexpressing Flag-tagged MPP9 (401–800 aa) and TTBK2-GFP-WT or TTBK2-GFP-KD after treatment with λ-PPase. Tubulin was used as a loading control. d Mass spectrometric analysis showing phosphorylation sites of MPP9 at S629 and S636. e Immunoblots of lysates from HEK293T cells co-overexpressing TTBK2-GFP and Flag-MPP9 (401–800 aa)-WT, or the indicated unphosphorylatable mutants. Tubulin was used as a loading control. f GST-MPP9 (401–800 aa)-WT, or the indicated mutants were subjected to a TTBK2 kinase assay in vitro followed by autoradiography. Coomassie blue (CBB) staining showed GST-tagged proteins. Relative amounts of phosphorylated MPP9 signals were quantified. g Lysates of HEK293T cells co-overexpressing Flag-tagged MPP9-WT or the S629A mutant and TTBK2-GFP were subjected to immunoprecipitation (IP) and immunoblotting with anti-Flag and anti-Phospho-S629 antibodies. Relative amounts of S629-phosphorylated MPP9 were quantified. h Immunostaining of S629-phosphorylated MPP9 (red) and Flag in Flag-MPP9 (green, upper) or Flag-CEP170 (green, lower) overexpressing hTERT RPE-1 cells. i Immunostaining of S629-phosphorylated MPP9 (red) and Flag in Flag-MPP9 (green) or Flag-MPP9–629A (green) overexpressing hTERT RPE-1 cells after depleting MPP9. j Quantifications of S629-phosphorylated MPP9 signals in i ( n = 50 cells for each group). k Immunostaining of S629-phosphorylated MPP9 (green) and Centrin-3 (red) in control- or TTBK2-siRNA transfected hTERT RPE-1 cells. l Quantifications of S629-phosphorylated MPP9 signals in k ( n = 50 cells for each group). m Lysates of control-siRNA or TTBK2-siRNA transfected hTERT RPE-1 cells after serum starvation were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. Relative amounts of S629-phosphorylated MPP9 were quantified and normalized to MPP9. For j , l , bars represent the means ± S.E.M for three independent experiments. *** p

    Techniques Used: Western Blot, Kinase Assay, In Vitro, Autoradiography, Staining, Mutagenesis, Immunoprecipitation, Immunostaining, Transfection

    2) Product Images from "NTP-mediated nucleotide excision activity of hepatitis C virus RNA-dependent RNA polymerase"

    Article Title: NTP-mediated nucleotide excision activity of hepatitis C virus RNA-dependent RNA polymerase

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

    doi: 10.1073/pnas.1214924110

    HCV polymerase (NS5B) catalyzes NTP-mediated excision of a 3′-terminal CMP from a nascent RNA primer. ( A ) Procedure to obtain a purified NS5B–10/20-mer elongation complex with α- 32 P-CMP (indicated as C ) at the 3′-end of the 10-mer primer. The sequence of the RNA template (20-mer) used in this study is shown. ( B ) Sequencing gel image shows excision of the α- 32 P-CMP from 10-mer RNA substrate by PP i or NTPs and generation of CTP or Np 4 C dinucleoside tetraphosphates. For each reaction, the purified NS5B–10/20-mer elongation complex (0.3 μM) was incubated for 7 min with 1 mM PP i or NTP, pyrophosphatase-treated (+) or untreated (−). Lane 1: NS5B–10/20-mer elongation complex before purification. rxn, reaction. Lane 2: purified elongation complex. As a position reference, dinucleoside tetraphosphates Ap 4 C, Up 4 C, Cp 4 C, and Gp 4 C synthesized by firefly luciferase were loaded in lanes 7, 10, 13, and 16, respectively, and their positions are indicated by arrows.
    Figure Legend Snippet: HCV polymerase (NS5B) catalyzes NTP-mediated excision of a 3′-terminal CMP from a nascent RNA primer. ( A ) Procedure to obtain a purified NS5B–10/20-mer elongation complex with α- 32 P-CMP (indicated as C ) at the 3′-end of the 10-mer primer. The sequence of the RNA template (20-mer) used in this study is shown. ( B ) Sequencing gel image shows excision of the α- 32 P-CMP from 10-mer RNA substrate by PP i or NTPs and generation of CTP or Np 4 C dinucleoside tetraphosphates. For each reaction, the purified NS5B–10/20-mer elongation complex (0.3 μM) was incubated for 7 min with 1 mM PP i or NTP, pyrophosphatase-treated (+) or untreated (−). Lane 1: NS5B–10/20-mer elongation complex before purification. rxn, reaction. Lane 2: purified elongation complex. As a position reference, dinucleoside tetraphosphates Ap 4 C, Up 4 C, Cp 4 C, and Gp 4 C synthesized by firefly luciferase were loaded in lanes 7, 10, 13, and 16, respectively, and their positions are indicated by arrows.

    Techniques Used: Purification, Sequencing, Incubation, Synthesized, Luciferase

    3) Product Images from "Reconstitution of polythioamide antibiotic backbone formation reveals unusual thiotemplated assembly strategy"

    Article Title: Reconstitution of polythioamide antibiotic backbone formation reveals unusual thiotemplated assembly strategy

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

    doi: 10.1073/pnas.1918759117

    CtaI loads PHBA onto holo -CtaH. ( A ) MALDI-TOF spectral overlay of holo -CtaH loading reactions performed with PHBA and either CtaI or CtaD. The expected average masses of holo -CtaH and PHBA-S-CtaH are indicated. ( B ) HPLC absorbance profiles (250 nm) of reactions performed with NAC, PHBA, and CtaI. ( C ) Quantification of ATP hydrolysis products using a malachite green assay. Error bars represent the SD from the mean ( n = 3). PPase, inorganic pyrophosphatase. ( D ) HPLC profiles of CtaI reactions with NAC. Red strikethrough indicates heat-inactivated enzyme; red asterisk, NAC-specific peak.
    Figure Legend Snippet: CtaI loads PHBA onto holo -CtaH. ( A ) MALDI-TOF spectral overlay of holo -CtaH loading reactions performed with PHBA and either CtaI or CtaD. The expected average masses of holo -CtaH and PHBA-S-CtaH are indicated. ( B ) HPLC absorbance profiles (250 nm) of reactions performed with NAC, PHBA, and CtaI. ( C ) Quantification of ATP hydrolysis products using a malachite green assay. Error bars represent the SD from the mean ( n = 3). PPase, inorganic pyrophosphatase. ( D ) HPLC profiles of CtaI reactions with NAC. Red strikethrough indicates heat-inactivated enzyme; red asterisk, NAC-specific peak.

    Techniques Used: High Performance Liquid Chromatography, Malachite Green Assay

    4) Product Images from "Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection"

    Article Title: Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1038/mtna.2014.66

    MicroRNA synthesized by rolling circle transcription (RCT) site-specific disconnection (SSD) with various concentrations of circular cDNA template . ( a ) Time course of RCT in the absence of inorganic pyrophosphatase (IPP). ( b ) Effect of IPP on the amount of RNA products. Lane L, 25 nt RNA marker; Lane S, standard sample of chemically synthesized mir-16 (1 μl, 100 μmol/l). In ( b ), RCT was processed for 4 hours, with 0.1 μmol/l Aid-DNA-16-1 and 1.0 U/ml IPP.
    Figure Legend Snippet: MicroRNA synthesized by rolling circle transcription (RCT) site-specific disconnection (SSD) with various concentrations of circular cDNA template . ( a ) Time course of RCT in the absence of inorganic pyrophosphatase (IPP). ( b ) Effect of IPP on the amount of RNA products. Lane L, 25 nt RNA marker; Lane S, standard sample of chemically synthesized mir-16 (1 μl, 100 μmol/l). In ( b ), RCT was processed for 4 hours, with 0.1 μmol/l Aid-DNA-16-1 and 1.0 U/ml IPP.

    Techniques Used: Synthesized, Marker

    Related Articles

    Clone Assay:

    Article Title: Multiplexed in vivo His-tagging of enzyme pathways for in vitro single-pot multi-enzyme catalysis
    Article Snippet: .. A typical 10 μL PURE reaction contained all 31 factors (IF1, IF2, IF3, EF-G, EF-Ts, EF-Tu, RF1, RF2, RF3, RRF, methionyl-tRNA formyl-transferase, and 20 aaRS), 20 μg/mL pyruvate kinase (Sigma), 3.0 μg/mL myokinase (EMD Chemical), 1.1 μg/mL nucleotide diphosphate kinase (Sigma), and 2 U/mL pyrophosphatase (New England Biolab), 10 μg/mL T7 RNA polymerase (Ambion), 0.1 mM of 20 amino acids (Sigma), 54 A260 unit of E. coli total tRNA (Roche), 2 mM ATP, 2 mM GTP, 1 mM CTP, 1mM UTP, 20 mM phosphoenolpyruvate, 10 μg/mL formyl donor, 50mM Hepes-KOH (pH 7.6), 100 mM potassium glutamate, 13 mM Mg(OAc)2 , 2 mM spermidine, 1mM DTT, 0.8 U/μL Murine RNase Inhibitor (New England Biolab), 1.2 μM of ribosome, and 10 μg/mL of the pIVEX-Luc plasmid in which the firefly luciferase gene under T7 regulation was cloned into plasmid pIVEX 2.3d (from 5-Prime). .. All ePURE translation reactions contained the same components as the PURE reaction except that the 31 factors are replaced by 700 μg/mL IEF (containing IF1, IF2, IF3, EF-G, EFTs, EF-Tu, EF-4), 370 μg/mL RF (containing RF1, RF2, RF3, RRF), 150 μg/mL RS1 (containing CysRS, GlnRS, IleRS, LeuRS, ProRS, SerRS), 550 μg/mL RS2 (containing AsnRS, AspRS, PheRS, ThrRS, TyrRS), 183 μg/mL RS3 (containing AlaRS, ArgRS, GluRS, HisRS, LysRS, MetRS), 79 μg/mL RS4 (containing GlyRS, TrpRS, ValRS, methionyl-tRNA formyl-transferase) and four supplementary, individually prepared factors: 22.3 μg/mL IF1, 30.9 μg/mL IF3, 20.0 μg/mL ArgRS and 96.0 μg/mL GlyRS.

    Luciferase:

    Article Title: NTP-mediated nucleotide excision activity of hepatitis C virus RNA-dependent RNA polymerase
    Article Snippet: .. In a 50-μL reaction, 20 μCi [α-32 P]CTP and one of the NTPs (ATP, GTP, UTP, or CTP) at 2 mM were incubated for 24 h at 30 °C with 25 μg of firefly luciferase (Sigma–Aldrich) in a reaction buffer containing 50 mM Hepes (pH 7.4), 5 mM MgCl2 , 1 mg/mL BSA, 0.05 units of yeast inorganic pyrophosphatase (PPase; New England Biolabs), and 0.1 mM D-Luciferin (Sigma–Aldrich). .. In a typical assay, purified NS5B–10/20-mer elongation complex was incubated at 30 °C with one NTP or PPi for time intervals as indicated.

    Article Title: Multiplexed in vivo His-tagging of enzyme pathways for in vitro single-pot multi-enzyme catalysis
    Article Snippet: .. A typical 10 μL PURE reaction contained all 31 factors (IF1, IF2, IF3, EF-G, EF-Ts, EF-Tu, RF1, RF2, RF3, RRF, methionyl-tRNA formyl-transferase, and 20 aaRS), 20 μg/mL pyruvate kinase (Sigma), 3.0 μg/mL myokinase (EMD Chemical), 1.1 μg/mL nucleotide diphosphate kinase (Sigma), and 2 U/mL pyrophosphatase (New England Biolab), 10 μg/mL T7 RNA polymerase (Ambion), 0.1 mM of 20 amino acids (Sigma), 54 A260 unit of E. coli total tRNA (Roche), 2 mM ATP, 2 mM GTP, 1 mM CTP, 1mM UTP, 20 mM phosphoenolpyruvate, 10 μg/mL formyl donor, 50mM Hepes-KOH (pH 7.6), 100 mM potassium glutamate, 13 mM Mg(OAc)2 , 2 mM spermidine, 1mM DTT, 0.8 U/μL Murine RNase Inhibitor (New England Biolab), 1.2 μM of ribosome, and 10 μg/mL of the pIVEX-Luc plasmid in which the firefly luciferase gene under T7 regulation was cloned into plasmid pIVEX 2.3d (from 5-Prime). .. All ePURE translation reactions contained the same components as the PURE reaction except that the 31 factors are replaced by 700 μg/mL IEF (containing IF1, IF2, IF3, EF-G, EFTs, EF-Tu, EF-4), 370 μg/mL RF (containing RF1, RF2, RF3, RRF), 150 μg/mL RS1 (containing CysRS, GlnRS, IleRS, LeuRS, ProRS, SerRS), 550 μg/mL RS2 (containing AsnRS, AspRS, PheRS, ThrRS, TyrRS), 183 μg/mL RS3 (containing AlaRS, ArgRS, GluRS, HisRS, LysRS, MetRS), 79 μg/mL RS4 (containing GlyRS, TrpRS, ValRS, methionyl-tRNA formyl-transferase) and four supplementary, individually prepared factors: 22.3 μg/mL IF1, 30.9 μg/mL IF3, 20.0 μg/mL ArgRS and 96.0 μg/mL GlyRS.

    In Vitro:

    Article Title: Structure of an Escherichia coli Hfq:RNA complex at 0.97 Å resolution
    Article Snippet: .. For in vitro transcription the purified DNA template (0.1 mg ml−1 ) was incubated with T7 RNA polymerase (0.2 mg ml−1 ) and 20 units of pyrophosphatase (NEB) in TXN buffer [0.02 M MgCl2 , 0.04 M of each rNTP, 0.3 M HEPES–NaOH pH 8.0, 0.005 M DTT, 0.001 M spermidine, 0.01%( v / v ) Triton X-100] at 37°C for 18 h. After ethanol precipitation of the transcript, cleavage of the 3′ ribozyme GlmS from the RNA transcript was induced by addition of 0.05 M GlcN6P. .. Finally, RNA-OUT was purified via ion-exchange chromatography on a Resource Q column.

    Mutagenesis:

    Article Title: Characterization of Enzymes from Legionella pneumophila Involved in Reversible Adenylylation of Rab1 Protein *
    Article Snippet: .. For all adenylylation experiments, the quadruple mutant DrrAfl (N451A/R453A/D480A/S483A) was used to exclude effects of the GEF activity of DrrA ( , ), and all measurements of DrrA-catalyzed adenylylation were performed in the presence of 2.5 units of yeast inorganic pyrophosphatase (New England Biolabs) to degrade pyrophosphate produced in the reaction. ..

    Ethanol Precipitation:

    Article Title: Structure of an Escherichia coli Hfq:RNA complex at 0.97 Å resolution
    Article Snippet: .. For in vitro transcription the purified DNA template (0.1 mg ml−1 ) was incubated with T7 RNA polymerase (0.2 mg ml−1 ) and 20 units of pyrophosphatase (NEB) in TXN buffer [0.02 M MgCl2 , 0.04 M of each rNTP, 0.3 M HEPES–NaOH pH 8.0, 0.005 M DTT, 0.001 M spermidine, 0.01%( v / v ) Triton X-100] at 37°C for 18 h. After ethanol precipitation of the transcript, cleavage of the 3′ ribozyme GlmS from the RNA transcript was induced by addition of 0.05 M GlcN6P. .. Finally, RNA-OUT was purified via ion-exchange chromatography on a Resource Q column.

    Purification:

    Article Title: Structure of an Escherichia coli Hfq:RNA complex at 0.97 Å resolution
    Article Snippet: .. For in vitro transcription the purified DNA template (0.1 mg ml−1 ) was incubated with T7 RNA polymerase (0.2 mg ml−1 ) and 20 units of pyrophosphatase (NEB) in TXN buffer [0.02 M MgCl2 , 0.04 M of each rNTP, 0.3 M HEPES–NaOH pH 8.0, 0.005 M DTT, 0.001 M spermidine, 0.01%( v / v ) Triton X-100] at 37°C for 18 h. After ethanol precipitation of the transcript, cleavage of the 3′ ribozyme GlmS from the RNA transcript was induced by addition of 0.05 M GlcN6P. .. Finally, RNA-OUT was purified via ion-exchange chromatography on a Resource Q column.

    Produced:

    Article Title: Characterization of Enzymes from Legionella pneumophila Involved in Reversible Adenylylation of Rab1 Protein *
    Article Snippet: .. For all adenylylation experiments, the quadruple mutant DrrAfl (N451A/R453A/D480A/S483A) was used to exclude effects of the GEF activity of DrrA ( , ), and all measurements of DrrA-catalyzed adenylylation were performed in the presence of 2.5 units of yeast inorganic pyrophosphatase (New England Biolabs) to degrade pyrophosphate produced in the reaction. ..

    Incubation:

    Article Title: NTP-mediated nucleotide excision activity of hepatitis C virus RNA-dependent RNA polymerase
    Article Snippet: .. In a 50-μL reaction, 20 μCi [α-32 P]CTP and one of the NTPs (ATP, GTP, UTP, or CTP) at 2 mM were incubated for 24 h at 30 °C with 25 μg of firefly luciferase (Sigma–Aldrich) in a reaction buffer containing 50 mM Hepes (pH 7.4), 5 mM MgCl2 , 1 mg/mL BSA, 0.05 units of yeast inorganic pyrophosphatase (PPase; New England Biolabs), and 0.1 mM D-Luciferin (Sigma–Aldrich). .. In a typical assay, purified NS5B–10/20-mer elongation complex was incubated at 30 °C with one NTP or PPi for time intervals as indicated.

    Article Title: Structure of an Escherichia coli Hfq:RNA complex at 0.97 Å resolution
    Article Snippet: .. For in vitro transcription the purified DNA template (0.1 mg ml−1 ) was incubated with T7 RNA polymerase (0.2 mg ml−1 ) and 20 units of pyrophosphatase (NEB) in TXN buffer [0.02 M MgCl2 , 0.04 M of each rNTP, 0.3 M HEPES–NaOH pH 8.0, 0.005 M DTT, 0.001 M spermidine, 0.01%( v / v ) Triton X-100] at 37°C for 18 h. After ethanol precipitation of the transcript, cleavage of the 3′ ribozyme GlmS from the RNA transcript was induced by addition of 0.05 M GlcN6P. .. Finally, RNA-OUT was purified via ion-exchange chromatography on a Resource Q column.

    Activity Assay:

    Article Title: Characterization of Enzymes from Legionella pneumophila Involved in Reversible Adenylylation of Rab1 Protein *
    Article Snippet: .. For all adenylylation experiments, the quadruple mutant DrrAfl (N451A/R453A/D480A/S483A) was used to exclude effects of the GEF activity of DrrA ( , ), and all measurements of DrrA-catalyzed adenylylation were performed in the presence of 2.5 units of yeast inorganic pyrophosphatase (New England Biolabs) to degrade pyrophosphate produced in the reaction. ..

    Plasmid Preparation:

    Article Title: Multiplexed in vivo His-tagging of enzyme pathways for in vitro single-pot multi-enzyme catalysis
    Article Snippet: .. A typical 10 μL PURE reaction contained all 31 factors (IF1, IF2, IF3, EF-G, EF-Ts, EF-Tu, RF1, RF2, RF3, RRF, methionyl-tRNA formyl-transferase, and 20 aaRS), 20 μg/mL pyruvate kinase (Sigma), 3.0 μg/mL myokinase (EMD Chemical), 1.1 μg/mL nucleotide diphosphate kinase (Sigma), and 2 U/mL pyrophosphatase (New England Biolab), 10 μg/mL T7 RNA polymerase (Ambion), 0.1 mM of 20 amino acids (Sigma), 54 A260 unit of E. coli total tRNA (Roche), 2 mM ATP, 2 mM GTP, 1 mM CTP, 1mM UTP, 20 mM phosphoenolpyruvate, 10 μg/mL formyl donor, 50mM Hepes-KOH (pH 7.6), 100 mM potassium glutamate, 13 mM Mg(OAc)2 , 2 mM spermidine, 1mM DTT, 0.8 U/μL Murine RNase Inhibitor (New England Biolab), 1.2 μM of ribosome, and 10 μg/mL of the pIVEX-Luc plasmid in which the firefly luciferase gene under T7 regulation was cloned into plasmid pIVEX 2.3d (from 5-Prime). .. All ePURE translation reactions contained the same components as the PURE reaction except that the 31 factors are replaced by 700 μg/mL IEF (containing IF1, IF2, IF3, EF-G, EFTs, EF-Tu, EF-4), 370 μg/mL RF (containing RF1, RF2, RF3, RRF), 150 μg/mL RS1 (containing CysRS, GlnRS, IleRS, LeuRS, ProRS, SerRS), 550 μg/mL RS2 (containing AsnRS, AspRS, PheRS, ThrRS, TyrRS), 183 μg/mL RS3 (containing AlaRS, ArgRS, GluRS, HisRS, LysRS, MetRS), 79 μg/mL RS4 (containing GlyRS, TrpRS, ValRS, methionyl-tRNA formyl-transferase) and four supplementary, individually prepared factors: 22.3 μg/mL IF1, 30.9 μg/mL IF3, 20.0 μg/mL ArgRS and 96.0 μg/mL GlyRS.

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication
    Article Snippet: .. Assay of the TTcDR reaction The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara). .. Most of the proteins described above were purified according to a previously described method except for yeast inorganic pyrophosphatase, ribonuclease inhibitor, and T7 RNA polymerase.

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    New England Biolabs ppase
    TTBK2 phosphorylates MPP9 mainly at S629 site. a Immunoblots of lysates from HEK293T cells co-overexpressing Flag-MPP9 and the indicated cilia-related kinase. Tubulin was used as a loading control. b Immunoblots of lysates from HEK293T cells co-overexpressing Flag-tagged MPP9 full-length (FL) or the indicated MPP9 truncation mutants with TTBK2-GFP-WT or TTBK2-GFP-KD. Tubulin was used as a loading control. WT, wild-type; KD, kinase-dead. c Immunoblots of lysates from HEK293T cells co-overexpressing Flag-tagged MPP9 (401–800 aa) and TTBK2-GFP-WT or TTBK2-GFP-KD after treatment with <t>λ-PPase.</t> Tubulin was used as a loading control. d Mass spectrometric analysis showing phosphorylation sites of MPP9 at S629 and S636. e Immunoblots of lysates from HEK293T cells co-overexpressing TTBK2-GFP and Flag-MPP9 (401–800 aa)-WT, or the indicated unphosphorylatable mutants. Tubulin was used as a loading control. f GST-MPP9 (401–800 aa)-WT, or the indicated mutants were subjected to a TTBK2 kinase assay in vitro followed by autoradiography. Coomassie blue (CBB) staining showed GST-tagged proteins. Relative amounts of phosphorylated MPP9 signals were quantified. g Lysates of HEK293T cells co-overexpressing Flag-tagged MPP9-WT or the S629A mutant and TTBK2-GFP were subjected to immunoprecipitation (IP) and immunoblotting with anti-Flag and anti-Phospho-S629 antibodies. Relative amounts of S629-phosphorylated MPP9 were quantified. h Immunostaining of S629-phosphorylated MPP9 (red) and Flag in Flag-MPP9 (green, upper) or Flag-CEP170 (green, lower) overexpressing hTERT RPE-1 cells. i Immunostaining of S629-phosphorylated MPP9 (red) and Flag in Flag-MPP9 (green) or Flag-MPP9–629A (green) overexpressing hTERT RPE-1 cells after depleting MPP9. j Quantifications of S629-phosphorylated MPP9 signals in i ( n = 50 cells for each group). k Immunostaining of S629-phosphorylated MPP9 (green) and Centrin-3 (red) in control- or TTBK2-siRNA transfected hTERT RPE-1 cells. l Quantifications of S629-phosphorylated MPP9 signals in k ( n = 50 cells for each group). m Lysates of control-siRNA or TTBK2-siRNA transfected hTERT RPE-1 cells after serum starvation were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. Relative amounts of S629-phosphorylated MPP9 were quantified and normalized to MPP9. For j , l , bars represent the means ± S.E.M for three independent experiments. *** p
    Ppase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ppase/product/New England Biolabs
    Average 98 stars, based on 33 article reviews
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    TTBK2 phosphorylates MPP9 mainly at S629 site. a Immunoblots of lysates from HEK293T cells co-overexpressing Flag-MPP9 and the indicated cilia-related kinase. Tubulin was used as a loading control. b Immunoblots of lysates from HEK293T cells co-overexpressing Flag-tagged MPP9 full-length (FL) or the indicated MPP9 truncation mutants with TTBK2-GFP-WT or TTBK2-GFP-KD. Tubulin was used as a loading control. WT, wild-type; KD, kinase-dead. c Immunoblots of lysates from HEK293T cells co-overexpressing Flag-tagged MPP9 (401–800 aa) and TTBK2-GFP-WT or TTBK2-GFP-KD after treatment with λ-PPase. Tubulin was used as a loading control. d Mass spectrometric analysis showing phosphorylation sites of MPP9 at S629 and S636. e Immunoblots of lysates from HEK293T cells co-overexpressing TTBK2-GFP and Flag-MPP9 (401–800 aa)-WT, or the indicated unphosphorylatable mutants. Tubulin was used as a loading control. f GST-MPP9 (401–800 aa)-WT, or the indicated mutants were subjected to a TTBK2 kinase assay in vitro followed by autoradiography. Coomassie blue (CBB) staining showed GST-tagged proteins. Relative amounts of phosphorylated MPP9 signals were quantified. g Lysates of HEK293T cells co-overexpressing Flag-tagged MPP9-WT or the S629A mutant and TTBK2-GFP were subjected to immunoprecipitation (IP) and immunoblotting with anti-Flag and anti-Phospho-S629 antibodies. Relative amounts of S629-phosphorylated MPP9 were quantified. h Immunostaining of S629-phosphorylated MPP9 (red) and Flag in Flag-MPP9 (green, upper) or Flag-CEP170 (green, lower) overexpressing hTERT RPE-1 cells. i Immunostaining of S629-phosphorylated MPP9 (red) and Flag in Flag-MPP9 (green) or Flag-MPP9–629A (green) overexpressing hTERT RPE-1 cells after depleting MPP9. j Quantifications of S629-phosphorylated MPP9 signals in i ( n = 50 cells for each group). k Immunostaining of S629-phosphorylated MPP9 (green) and Centrin-3 (red) in control- or TTBK2-siRNA transfected hTERT RPE-1 cells. l Quantifications of S629-phosphorylated MPP9 signals in k ( n = 50 cells for each group). m Lysates of control-siRNA or TTBK2-siRNA transfected hTERT RPE-1 cells after serum starvation were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. Relative amounts of S629-phosphorylated MPP9 were quantified and normalized to MPP9. For j , l , bars represent the means ± S.E.M for three independent experiments. *** p

    Journal: Nature Communications

    Article Title: M-Phase Phosphoprotein 9 regulates ciliogenesis by modulating CP110-CEP97 complex localization at the mother centriole

    doi: 10.1038/s41467-018-06990-9

    Figure Lengend Snippet: TTBK2 phosphorylates MPP9 mainly at S629 site. a Immunoblots of lysates from HEK293T cells co-overexpressing Flag-MPP9 and the indicated cilia-related kinase. Tubulin was used as a loading control. b Immunoblots of lysates from HEK293T cells co-overexpressing Flag-tagged MPP9 full-length (FL) or the indicated MPP9 truncation mutants with TTBK2-GFP-WT or TTBK2-GFP-KD. Tubulin was used as a loading control. WT, wild-type; KD, kinase-dead. c Immunoblots of lysates from HEK293T cells co-overexpressing Flag-tagged MPP9 (401–800 aa) and TTBK2-GFP-WT or TTBK2-GFP-KD after treatment with λ-PPase. Tubulin was used as a loading control. d Mass spectrometric analysis showing phosphorylation sites of MPP9 at S629 and S636. e Immunoblots of lysates from HEK293T cells co-overexpressing TTBK2-GFP and Flag-MPP9 (401–800 aa)-WT, or the indicated unphosphorylatable mutants. Tubulin was used as a loading control. f GST-MPP9 (401–800 aa)-WT, or the indicated mutants were subjected to a TTBK2 kinase assay in vitro followed by autoradiography. Coomassie blue (CBB) staining showed GST-tagged proteins. Relative amounts of phosphorylated MPP9 signals were quantified. g Lysates of HEK293T cells co-overexpressing Flag-tagged MPP9-WT or the S629A mutant and TTBK2-GFP were subjected to immunoprecipitation (IP) and immunoblotting with anti-Flag and anti-Phospho-S629 antibodies. Relative amounts of S629-phosphorylated MPP9 were quantified. h Immunostaining of S629-phosphorylated MPP9 (red) and Flag in Flag-MPP9 (green, upper) or Flag-CEP170 (green, lower) overexpressing hTERT RPE-1 cells. i Immunostaining of S629-phosphorylated MPP9 (red) and Flag in Flag-MPP9 (green) or Flag-MPP9–629A (green) overexpressing hTERT RPE-1 cells after depleting MPP9. j Quantifications of S629-phosphorylated MPP9 signals in i ( n = 50 cells for each group). k Immunostaining of S629-phosphorylated MPP9 (green) and Centrin-3 (red) in control- or TTBK2-siRNA transfected hTERT RPE-1 cells. l Quantifications of S629-phosphorylated MPP9 signals in k ( n = 50 cells for each group). m Lysates of control-siRNA or TTBK2-siRNA transfected hTERT RPE-1 cells after serum starvation were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. Relative amounts of S629-phosphorylated MPP9 were quantified and normalized to MPP9. For j , l , bars represent the means ± S.E.M for three independent experiments. *** p

    Article Snippet: For dephosphorylation assays, cell lysates were subjected to λ PPase (NEB) for 30 min, as based on the manufacturer’s protocols.

    Techniques: Western Blot, Kinase Assay, In Vitro, Autoradiography, Staining, Mutagenesis, Immunoprecipitation, Immunostaining, Transfection

    HCV polymerase (NS5B) catalyzes NTP-mediated excision of a 3′-terminal CMP from a nascent RNA primer. ( A ) Procedure to obtain a purified NS5B–10/20-mer elongation complex with α- 32 P-CMP (indicated as C ) at the 3′-end of the 10-mer primer. The sequence of the RNA template (20-mer) used in this study is shown. ( B ) Sequencing gel image shows excision of the α- 32 P-CMP from 10-mer RNA substrate by PP i or NTPs and generation of CTP or Np 4 C dinucleoside tetraphosphates. For each reaction, the purified NS5B–10/20-mer elongation complex (0.3 μM) was incubated for 7 min with 1 mM PP i or NTP, pyrophosphatase-treated (+) or untreated (−). Lane 1: NS5B–10/20-mer elongation complex before purification. rxn, reaction. Lane 2: purified elongation complex. As a position reference, dinucleoside tetraphosphates Ap 4 C, Up 4 C, Cp 4 C, and Gp 4 C synthesized by firefly luciferase were loaded in lanes 7, 10, 13, and 16, respectively, and their positions are indicated by arrows.

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

    Article Title: NTP-mediated nucleotide excision activity of hepatitis C virus RNA-dependent RNA polymerase

    doi: 10.1073/pnas.1214924110

    Figure Lengend Snippet: HCV polymerase (NS5B) catalyzes NTP-mediated excision of a 3′-terminal CMP from a nascent RNA primer. ( A ) Procedure to obtain a purified NS5B–10/20-mer elongation complex with α- 32 P-CMP (indicated as C ) at the 3′-end of the 10-mer primer. The sequence of the RNA template (20-mer) used in this study is shown. ( B ) Sequencing gel image shows excision of the α- 32 P-CMP from 10-mer RNA substrate by PP i or NTPs and generation of CTP or Np 4 C dinucleoside tetraphosphates. For each reaction, the purified NS5B–10/20-mer elongation complex (0.3 μM) was incubated for 7 min with 1 mM PP i or NTP, pyrophosphatase-treated (+) or untreated (−). Lane 1: NS5B–10/20-mer elongation complex before purification. rxn, reaction. Lane 2: purified elongation complex. As a position reference, dinucleoside tetraphosphates Ap 4 C, Up 4 C, Cp 4 C, and Gp 4 C synthesized by firefly luciferase were loaded in lanes 7, 10, 13, and 16, respectively, and their positions are indicated by arrows.

    Article Snippet: In a 50-μL reaction, 20 μCi [α-32 P]CTP and one of the NTPs (ATP, GTP, UTP, or CTP) at 2 mM were incubated for 24 h at 30 °C with 25 μg of firefly luciferase (Sigma–Aldrich) in a reaction buffer containing 50 mM Hepes (pH 7.4), 5 mM MgCl2 , 1 mg/mL BSA, 0.05 units of yeast inorganic pyrophosphatase (PPase; New England Biolabs), and 0.1 mM D-Luciferin (Sigma–Aldrich).

    Techniques: Purification, Sequencing, Incubation, Synthesized, Luciferase

    CtaI loads PHBA onto holo -CtaH. ( A ) MALDI-TOF spectral overlay of holo -CtaH loading reactions performed with PHBA and either CtaI or CtaD. The expected average masses of holo -CtaH and PHBA-S-CtaH are indicated. ( B ) HPLC absorbance profiles (250 nm) of reactions performed with NAC, PHBA, and CtaI. ( C ) Quantification of ATP hydrolysis products using a malachite green assay. Error bars represent the SD from the mean ( n = 3). PPase, inorganic pyrophosphatase. ( D ) HPLC profiles of CtaI reactions with NAC. Red strikethrough indicates heat-inactivated enzyme; red asterisk, NAC-specific peak.

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

    Article Title: Reconstitution of polythioamide antibiotic backbone formation reveals unusual thiotemplated assembly strategy

    doi: 10.1073/pnas.1918759117

    Figure Lengend Snippet: CtaI loads PHBA onto holo -CtaH. ( A ) MALDI-TOF spectral overlay of holo -CtaH loading reactions performed with PHBA and either CtaI or CtaD. The expected average masses of holo -CtaH and PHBA-S-CtaH are indicated. ( B ) HPLC absorbance profiles (250 nm) of reactions performed with NAC, PHBA, and CtaI. ( C ) Quantification of ATP hydrolysis products using a malachite green assay. Error bars represent the SD from the mean ( n = 3). PPase, inorganic pyrophosphatase. ( D ) HPLC profiles of CtaI reactions with NAC. Red strikethrough indicates heat-inactivated enzyme; red asterisk, NAC-specific peak.

    Article Snippet: Where indicated, 0.01 U of inorganic pyrophosphatase (New England BioLabs) was added to the reaction mixture.

    Techniques: High Performance Liquid Chromatography, Malachite Green Assay

    MicroRNA synthesized by rolling circle transcription (RCT) site-specific disconnection (SSD) with various concentrations of circular cDNA template . ( a ) Time course of RCT in the absence of inorganic pyrophosphatase (IPP). ( b ) Effect of IPP on the amount of RNA products. Lane L, 25 nt RNA marker; Lane S, standard sample of chemically synthesized mir-16 (1 μl, 100 μmol/l). In ( b ), RCT was processed for 4 hours, with 0.1 μmol/l Aid-DNA-16-1 and 1.0 U/ml IPP.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection

    doi: 10.1038/mtna.2014.66

    Figure Lengend Snippet: MicroRNA synthesized by rolling circle transcription (RCT) site-specific disconnection (SSD) with various concentrations of circular cDNA template . ( a ) Time course of RCT in the absence of inorganic pyrophosphatase (IPP). ( b ) Effect of IPP on the amount of RNA products. Lane L, 25 nt RNA marker; Lane S, standard sample of chemically synthesized mir-16 (1 μl, 100 μmol/l). In ( b ), RCT was processed for 4 hours, with 0.1 μmol/l Aid-DNA-16-1 and 1.0 U/ml IPP.

    Article Snippet: Inorganic pyrophosphatase (0.02 U) purchased from New England Biolabs, was applied to selected RCT reactions.

    Techniques: Synthesized, Marker