atp  (New England Biolabs)


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  • 99
    Name:
    Apyrase
    Description:
    Apyrase 50 units
    Catalog Number:
    M0398L
    Price:
    288
    Size:
    50 units
    Category:
    Other Enzymes
    Score:
    85
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    Structured Review

    New England Biolabs atp
    Apyrase
    Apyrase 50 units
    https://www.bioz.com/result/atp/product/New England Biolabs
    Average 99 stars, based on 0 article reviews
    Price from $9.99 to $1999.99
    atp - by Bioz Stars, 2019-12
    99/100 stars

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    Images

    1) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    2) Product Images from "Mechanosensitive pannexin-1 channels mediate microvascular metastatic cell survival"

    Article Title: Mechanosensitive pannexin-1 channels mediate microvascular metastatic cell survival

    Journal:

    doi: 10.1038/ncb3194

    Mechanosensitive PANX1 channels release ATP to increase cancer cell survival during intravascular membrane stretch
    Figure Legend Snippet: Mechanosensitive PANX1 channels release ATP to increase cancer cell survival during intravascular membrane stretch

    Techniques Used:

    PANX1 1–89 augments PANX1-mediated ATP release in metastatic breast cancer cells
    Figure Legend Snippet: PANX1 1–89 augments PANX1-mediated ATP release in metastatic breast cancer cells

    Techniques Used:

    3) Product Images from "ZCF32, a fungus specific Zn(II)2 Cys6 transcription factor, is a repressor of the biofilm development in the human pathogen Candida albicans"

    Article Title: ZCF32, a fungus specific Zn(II)2 Cys6 transcription factor, is a repressor of the biofilm development in the human pathogen Candida albicans

    Journal: Scientific Reports

    doi: 10.1038/srep31124

    SELEX revealed the sequence-specific binding of Zcf32 to double-stranded DNA. ( A ) An electrophoretic mobility shift assay (EMSA) was performed using purified Zcf32 ZFN-MBP fusion protein (with increasing protein concentrations as indicated) and ATP-[γ- 32 P] labelled 18 bp double-stranded DNA oligonucleotides 1 and 2 (O1- TACCCGATATAGCCGATG and O2- CCGATATAGCCGATGCAT). The mobility shift is illustrated by an asterisk. ( B ) SELEX revealed that Zcf32 binds to eighteen independent double-stranded DNA sequences. A consensus analysis was carried out for these DNA sequences using MEME analysis tool and a 9 bp long consensus logo was obtained. Below the logo, sequences of the 18 bp long ds DNA oligonucleotides O1, O2 and the variant V1 are shown wherein the consensus region is underlined. In the case of the variant oligonucleotides, the mutated base is highlighted in red colour. ( C ) An EMSA was carried out with oligonucleotide O1and variant oligonucleotides (V1 to V27) to analyze the critical positions in the binding consensus of Zcf32. The binding ability of recombinant Zcf32 ZFN-MBP protein was analyzed for 27 variant oligonucleotides. Two representative EMSA gels for O1 and variant oligonucleotides V1 to V9 are shown. Asterisk indicates the shift in the mobility of ATP-[γ- 32 P] labelled ds DNA. The second lane for each oligonucleotide sequence in the EMSA gel indicates the probe (ATP-[γ- 32 P] labelled ds DNA) control. ( D ) Summary of the results from the mutational analysis of Zcf32 binding consensus sequence is represented in the form of a bar graph. The intensity of the shifted DNA was measured using Multigauge tool for each reaction carried out. The intensity of the shifted band obtained from variant DNA oligonucleotide was divided by the same of O1 oligonucleotide, thus giving the fraction of protein bound to the particular oligonucleotide sequence. Finally, the data is presented as percent binding of Zcf32 ZFN-MBP to O1and variant oligonucleotides in the form of a bar graph. Mutational analysis with single base pair change at a particular position shows that T, A and C at positions 6, 7 and 9 respectively are critical for the binding of Zcf32 ZFN domain.
    Figure Legend Snippet: SELEX revealed the sequence-specific binding of Zcf32 to double-stranded DNA. ( A ) An electrophoretic mobility shift assay (EMSA) was performed using purified Zcf32 ZFN-MBP fusion protein (with increasing protein concentrations as indicated) and ATP-[γ- 32 P] labelled 18 bp double-stranded DNA oligonucleotides 1 and 2 (O1- TACCCGATATAGCCGATG and O2- CCGATATAGCCGATGCAT). The mobility shift is illustrated by an asterisk. ( B ) SELEX revealed that Zcf32 binds to eighteen independent double-stranded DNA sequences. A consensus analysis was carried out for these DNA sequences using MEME analysis tool and a 9 bp long consensus logo was obtained. Below the logo, sequences of the 18 bp long ds DNA oligonucleotides O1, O2 and the variant V1 are shown wherein the consensus region is underlined. In the case of the variant oligonucleotides, the mutated base is highlighted in red colour. ( C ) An EMSA was carried out with oligonucleotide O1and variant oligonucleotides (V1 to V27) to analyze the critical positions in the binding consensus of Zcf32. The binding ability of recombinant Zcf32 ZFN-MBP protein was analyzed for 27 variant oligonucleotides. Two representative EMSA gels for O1 and variant oligonucleotides V1 to V9 are shown. Asterisk indicates the shift in the mobility of ATP-[γ- 32 P] labelled ds DNA. The second lane for each oligonucleotide sequence in the EMSA gel indicates the probe (ATP-[γ- 32 P] labelled ds DNA) control. ( D ) Summary of the results from the mutational analysis of Zcf32 binding consensus sequence is represented in the form of a bar graph. The intensity of the shifted DNA was measured using Multigauge tool for each reaction carried out. The intensity of the shifted band obtained from variant DNA oligonucleotide was divided by the same of O1 oligonucleotide, thus giving the fraction of protein bound to the particular oligonucleotide sequence. Finally, the data is presented as percent binding of Zcf32 ZFN-MBP to O1and variant oligonucleotides in the form of a bar graph. Mutational analysis with single base pair change at a particular position shows that T, A and C at positions 6, 7 and 9 respectively are critical for the binding of Zcf32 ZFN domain.

    Techniques Used: Sequencing, Binding Assay, Electrophoretic Mobility Shift Assay, Purification, Mobility Shift, Variant Assay, Recombinant

    4) Product Images from "Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions"

    Article Title: Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions

    Journal: Nature Communications

    doi: 10.1038/ncomms10607

    Msh2–Msh3 scans DNA via one-dimensional (1D) sliding. ( a ) Representative traces of diffusing Msh2–Msh3 molecules with 1 mM of the indicated nucleotide and 50 mM NaCl in the imaging buffer (black: ADP; blue: ATP; orange: ATP–Mg +2 ; green: AMP–PNP; pink: no nucleotide). ( b ) The trajectories in a were used to calculate mean squared displacements (MSD) and the MSDs for each molecule were used to obtain an apparent 1D diffusion coefficient (black: ADP; blue: ATP; orange: ATP-Mg +2 ; green: AMP-PNP; pink: no nucleotide). Solid lines indicate linear fits through the MSD points. ( c ) Diffusion coefficients for at least 50 molecules in each nucleotide state (with 50 mM NaCl). Red diamonds indicate the mean of the distribution. * P value
    Figure Legend Snippet: Msh2–Msh3 scans DNA via one-dimensional (1D) sliding. ( a ) Representative traces of diffusing Msh2–Msh3 molecules with 1 mM of the indicated nucleotide and 50 mM NaCl in the imaging buffer (black: ADP; blue: ATP; orange: ATP–Mg +2 ; green: AMP–PNP; pink: no nucleotide). ( b ) The trajectories in a were used to calculate mean squared displacements (MSD) and the MSDs for each molecule were used to obtain an apparent 1D diffusion coefficient (black: ADP; blue: ATP; orange: ATP-Mg +2 ; green: AMP-PNP; pink: no nucleotide). Solid lines indicate linear fits through the MSD points. ( c ) Diffusion coefficients for at least 50 molecules in each nucleotide state (with 50 mM NaCl). Red diamonds indicate the mean of the distribution. * P value

    Techniques Used: Imaging, Diffusion-based Assay

    5) Product Images from "Establishment of an in vitro trans-splicing system in Trypanosoma brucei that requires endogenous spliced leader RNA"

    Article Title: Establishment of an in vitro trans-splicing system in Trypanosoma brucei that requires endogenous spliced leader RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq065

    The trans -splicing reaction is heat-inactivated, requires ATP and is time-dependent. ( A ) Trans- splicing in heat-inactivated extracts. For heat inactivation, the trans -splicing extract was incubated for 30 min at 55°C. The trans -splicing product was compared to that produced in untreated extract. Total RNA was prepared from the reaction and subjected to RNAse protection as described in ‘Materials and Methods’ section. Lane contents are as follows: 1, RNase protection assay with RNA (30 µg) from pNS21-TIR transgenic cells; 2, In vitro trans- splicing reaction using untreated extract (control); 3, In vitro trans- splicing reaction using heated-inactivated extract. ( B ) Co-transcriptional trans- splicing and ATP requirement of the trans -splicing reaction. In vitro trans- splicing was performed in the presence of 0.8 µg pNS-21-TIR plasmid DNA as described in the ‘Results’ section. For ATP depletion, the ATP regenerating system was omitted from the reaction. After incubation at 28°C for 1 h, total RNA was prepared and subjected to RNase protection as described in ‘Materials and Methods’ section. Lane contents are as follows: 1, Antisense probe (500 c.p.m.); 2, total RNA (30 µg) from the transgenic pNS21-TIR cells; 3, RNA from extracts containing plasmid pNS21-TIR; 4, RNA from trans -splicing reaction lacking the ATP regenerating system; 5, RNA from trans -splicing performed under optimal conditions (control). M- DNA marker, labeled pBR322 DNA Msp I digest. ( C ) Kinetics of the in vitro trans- splicing reaction. In vitro trans -splicing was conducted under standard conditions but for different durations (10′, 60′ and 120′). RNA was subjected to RNase protection as described in ‘Materials and Methods’ section. ( D ) Quantitation of the time-dependent splicing from three independent experiments.
    Figure Legend Snippet: The trans -splicing reaction is heat-inactivated, requires ATP and is time-dependent. ( A ) Trans- splicing in heat-inactivated extracts. For heat inactivation, the trans -splicing extract was incubated for 30 min at 55°C. The trans -splicing product was compared to that produced in untreated extract. Total RNA was prepared from the reaction and subjected to RNAse protection as described in ‘Materials and Methods’ section. Lane contents are as follows: 1, RNase protection assay with RNA (30 µg) from pNS21-TIR transgenic cells; 2, In vitro trans- splicing reaction using untreated extract (control); 3, In vitro trans- splicing reaction using heated-inactivated extract. ( B ) Co-transcriptional trans- splicing and ATP requirement of the trans -splicing reaction. In vitro trans- splicing was performed in the presence of 0.8 µg pNS-21-TIR plasmid DNA as described in the ‘Results’ section. For ATP depletion, the ATP regenerating system was omitted from the reaction. After incubation at 28°C for 1 h, total RNA was prepared and subjected to RNase protection as described in ‘Materials and Methods’ section. Lane contents are as follows: 1, Antisense probe (500 c.p.m.); 2, total RNA (30 µg) from the transgenic pNS21-TIR cells; 3, RNA from extracts containing plasmid pNS21-TIR; 4, RNA from trans -splicing reaction lacking the ATP regenerating system; 5, RNA from trans -splicing performed under optimal conditions (control). M- DNA marker, labeled pBR322 DNA Msp I digest. ( C ) Kinetics of the in vitro trans- splicing reaction. In vitro trans -splicing was conducted under standard conditions but for different durations (10′, 60′ and 120′). RNA was subjected to RNase protection as described in ‘Materials and Methods’ section. ( D ) Quantitation of the time-dependent splicing from three independent experiments.

    Techniques Used: Incubation, Produced, Rnase Protection Assay, Transgenic Assay, In Vitro, Plasmid Preparation, Marker, Labeling, Quantitation Assay

    Detection of the trans- spliced product by RT-PCR and real-time PCR. ( A ) RT–PCR assay of the in vitro trans -splicing reaction. RNA was prepared from transgenic cells expressing pNS21-TIRsub and from in vitro trans -splicing assays using pre-mRNA of TIR-sub. cDNA was prepared from the different samples and was subjected to amplification with the different probes (pre-mRNA, tubulin and the primers specific for pNS21-TIRsub), as described in ‘Materials and Methods’ section. The primers are listed in S-1. The PCR product was separated on a 10% acrylamide gel and stained with ethidium bromide. The in vitro reaction was performed in the absence of ATP regenerating system or using heat-inactivated extract, prepared as described in Figure 3 . Lane contents are as follows: 1, cDNA from cell line expressing pNS21-TIRsub; 2, cDNA prepared from in vitro reaction performed under standard conditions; 3, cDNA prepared from in vitro trans -splicing performed in the absence of ATP regenerating system; 4, cDNA prepared from in vitro trans -splicing using a heat-inactivated extract. ( B ) RNase protection assay. RNA prepared from the in vitro reactions performed in Panel A was subjected to RNase protection assay. The RNA was separated on a 6% denaturing gel. Lane contents are as follows: 1, RNA from in vitro trans -splicing using pre-TIRsub; 2, the same as in lane 1 but the in vitro reaction lacked ATP regenerating system; 3, the same as in lane 1 but a heat-inactivated extract was used. The identity of the fragments is indicated. ( C ) Quantitation of the real-time PCR reactions. Real time-PCR was performed as described in ‘Materials and Methods’ section. The concentration curve was used to determine the total amount of PCR amplified in each reaction. The results shown are the average of three independent experiments. The designation of the different columns are: in vivo , the amount of PCR product (fmol) from 10 µg total RNA extracted from the transgenic cell line; IVTS, amount of PCR product (fmol) produced from a standard in vitro trans -splicing reaction; – ATP, amount of PCR product (fmol) produced from an in vitro trans -splicing reaction in the absence of the ATP regeneration system; heat inactivation, amount of PCR product (fmol) produced from one in vitro trans -splicing reaction using heat-inactivated extract.
    Figure Legend Snippet: Detection of the trans- spliced product by RT-PCR and real-time PCR. ( A ) RT–PCR assay of the in vitro trans -splicing reaction. RNA was prepared from transgenic cells expressing pNS21-TIRsub and from in vitro trans -splicing assays using pre-mRNA of TIR-sub. cDNA was prepared from the different samples and was subjected to amplification with the different probes (pre-mRNA, tubulin and the primers specific for pNS21-TIRsub), as described in ‘Materials and Methods’ section. The primers are listed in S-1. The PCR product was separated on a 10% acrylamide gel and stained with ethidium bromide. The in vitro reaction was performed in the absence of ATP regenerating system or using heat-inactivated extract, prepared as described in Figure 3 . Lane contents are as follows: 1, cDNA from cell line expressing pNS21-TIRsub; 2, cDNA prepared from in vitro reaction performed under standard conditions; 3, cDNA prepared from in vitro trans -splicing performed in the absence of ATP regenerating system; 4, cDNA prepared from in vitro trans -splicing using a heat-inactivated extract. ( B ) RNase protection assay. RNA prepared from the in vitro reactions performed in Panel A was subjected to RNase protection assay. The RNA was separated on a 6% denaturing gel. Lane contents are as follows: 1, RNA from in vitro trans -splicing using pre-TIRsub; 2, the same as in lane 1 but the in vitro reaction lacked ATP regenerating system; 3, the same as in lane 1 but a heat-inactivated extract was used. The identity of the fragments is indicated. ( C ) Quantitation of the real-time PCR reactions. Real time-PCR was performed as described in ‘Materials and Methods’ section. The concentration curve was used to determine the total amount of PCR amplified in each reaction. The results shown are the average of three independent experiments. The designation of the different columns are: in vivo , the amount of PCR product (fmol) from 10 µg total RNA extracted from the transgenic cell line; IVTS, amount of PCR product (fmol) produced from a standard in vitro trans -splicing reaction; – ATP, amount of PCR product (fmol) produced from an in vitro trans -splicing reaction in the absence of the ATP regeneration system; heat inactivation, amount of PCR product (fmol) produced from one in vitro trans -splicing reaction using heat-inactivated extract.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, In Vitro, Transgenic Assay, Expressing, Amplification, Polymerase Chain Reaction, Acrylamide Gel Assay, Staining, Rnase Protection Assay, Quantitation Assay, Concentration Assay, In Vivo, Produced

    6) Product Images from "Specific phosphorylation of the PfRh2b invasion ligand of Plasmodium falciparum"

    Article Title: Specific phosphorylation of the PfRh2b invasion ligand of Plasmodium falciparum

    Journal: Biochemical Journal

    doi: 10.1042/BJ20121694

    Inhibition of in vitro phosphorylation ( A ) Recombinant Rh2b was used in in vitro phosphorylation assays in the presence of [γ- 32 P]ATP with parasite lysate and different concentrations of the known CK2 inhibitor heparin. Phosphorylation as reduced by 1 μg/ml and eliminated by 5 and 10 μg/ml heparin. Upper panel, autoradiography. Lower panel, Coomassie Blue-stained SDS/PAGE. ( B ) The kinase, mediating Rh2b phosphorylation, was able to use ATP as well as GTP as phosphate donors. Rh2b CPD was not phosphorylated by parasite lysate in the absence of a phosphate donor. Upper panel, detection of phosphorylated Rh2b using the phospho-specific anti-pRh2b antibody. Lower panel, anti-GST antibody was used as a loading control. ( C ) The known CK2-specific inhibitor TBCA inhibited Rh2b CPD phosphorylation in a dose-dependent manner. In presence of parasite lysate, 10 μM TBCA reduced Rh2b phosphorylation, whereas 50 μM TBCA entirely eliminated phosphorylation. The solvent (DMSO) had no effect on Rh2b phosphorylation. Upper panel, autoradiography. Lower panel, Coomassie Blue-stained SDS/PAGE. ( D ) Phosphorylation of Rh2b CPD by parasite lysate was resistant to staurosporine (STP) treatment. Whereas cAMP-dependent AMA1 phosphorylation was inhibited by 1 μM staurosporine, Rh2b phosphorylation was resistant and only affected by higher doses. Upper panel, autoradiography. Lower panel, Coomassie Blue-stained SDS/PAGE. The molecular mass is shown in kDa on the left-hand side of the blots.
    Figure Legend Snippet: Inhibition of in vitro phosphorylation ( A ) Recombinant Rh2b was used in in vitro phosphorylation assays in the presence of [γ- 32 P]ATP with parasite lysate and different concentrations of the known CK2 inhibitor heparin. Phosphorylation as reduced by 1 μg/ml and eliminated by 5 and 10 μg/ml heparin. Upper panel, autoradiography. Lower panel, Coomassie Blue-stained SDS/PAGE. ( B ) The kinase, mediating Rh2b phosphorylation, was able to use ATP as well as GTP as phosphate donors. Rh2b CPD was not phosphorylated by parasite lysate in the absence of a phosphate donor. Upper panel, detection of phosphorylated Rh2b using the phospho-specific anti-pRh2b antibody. Lower panel, anti-GST antibody was used as a loading control. ( C ) The known CK2-specific inhibitor TBCA inhibited Rh2b CPD phosphorylation in a dose-dependent manner. In presence of parasite lysate, 10 μM TBCA reduced Rh2b phosphorylation, whereas 50 μM TBCA entirely eliminated phosphorylation. The solvent (DMSO) had no effect on Rh2b phosphorylation. Upper panel, autoradiography. Lower panel, Coomassie Blue-stained SDS/PAGE. ( D ) Phosphorylation of Rh2b CPD by parasite lysate was resistant to staurosporine (STP) treatment. Whereas cAMP-dependent AMA1 phosphorylation was inhibited by 1 μM staurosporine, Rh2b phosphorylation was resistant and only affected by higher doses. Upper panel, autoradiography. Lower panel, Coomassie Blue-stained SDS/PAGE. The molecular mass is shown in kDa on the left-hand side of the blots.

    Techniques Used: Inhibition, In Vitro, Recombinant, Autoradiography, Staining, SDS Page

    7) Product Images from "Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)"

    Article Title: Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)

    Journal: International Journal of Plant Genomics

    doi: 10.1155/2009/915061

    Synthesis and ligation of high efficiency 3′ adenylated cloning linkers. (a) An adenosine 5′-phosphorimidazolide is attached, in the presence of magnesium chloride, to a synthetic deoxyribo-oligonucleotide bearing a dideoxycytidine (ddC) block on its 3′ end and a free, reactive phosphate group on its 5′ end. (b) The synthetic, preactivated 3′ linker is ligated to target small RNAs in the presence of T4 RNA Ligase. This reaction is carried out with high efficiency in the absence of ATP to prevent circularization of the target RNA species prior to ligation. Reaction energy is provided by the phosphorimidazolide at the 5′ end of the linker.
    Figure Legend Snippet: Synthesis and ligation of high efficiency 3′ adenylated cloning linkers. (a) An adenosine 5′-phosphorimidazolide is attached, in the presence of magnesium chloride, to a synthetic deoxyribo-oligonucleotide bearing a dideoxycytidine (ddC) block on its 3′ end and a free, reactive phosphate group on its 5′ end. (b) The synthetic, preactivated 3′ linker is ligated to target small RNAs in the presence of T4 RNA Ligase. This reaction is carried out with high efficiency in the absence of ATP to prevent circularization of the target RNA species prior to ligation. Reaction energy is provided by the phosphorimidazolide at the 5′ end of the linker.

    Techniques Used: Ligation, Clone Assay, Blocking Assay

    8) Product Images from "The Carboxyl Terminus of Brca2 Links the Disassembly of Rad51 Complexes to Mitotic Entry"

    Article Title: The Carboxyl Terminus of Brca2 Links the Disassembly of Rad51 Complexes to Mitotic Entry

    Journal: Current Biology

    doi: 10.1016/j.cub.2009.05.057

    The C-Terminal Motif of Gg Brca2 Is Functionally Cognate with Its Human Counterpart in CDK-Regulated Rad51 Binding (A) Protein sequence alignment showing the conserved cyclin-dependent kinase (CDK)-phosphorylated residues flanking Ser3239 of Gg Brca 2 and Ser3291 of Hs BRCA2. Gallus gallus residues used in this study are boxed, and residue numbers are indicated below. Asterisks indicate identical residues, double dots indicate conserved substitutions, and single dots indicate residues that are semiconserved. (B) Western blot analysis of Gg Rad51 and the myc epitope following immunoprecipitation of the myc-tagged construct encoding residues 3152–3398 of Gg Brca2 ( Gg B2 3152 aa–3398 aa ) transfected into DT40 cells. Roscovitine treatment for 30–120 min in asynchronous cells had little effect on Gg Rad51 binding (lanes 2 and 3). However, roscovitine effectively reversed the reduction in binding induced by nocodazole (compare lanes 5 and 6 with lane 4). (C and D) Western blot analysis of Gg Rad51 and the myc epitope following immunoprecipitation comparing the wild-type (WT) with S3239A/E or P3240L (C) or T3232A (D) variants of Gg B2 3152 aa–3398 aa transfected into DT40 cells. The S3239A/E and P3240L mutations in the conserved Ser-Pro consensus for CDK phosphorylation abrogated binding to Gg Rad51 in asynchronous and nocodazole-arrested mitotic cell extracts, whereas the T3232A mutation caused enhanced binding to Gg Rad51 under the same conditions. (E) Thr3232 and Ser3239 can be phosphorylated in vitro by CDK1. A wild-type Gg Brca2 peptide fused with GST (WT, sequence at top) or mutant forms in which Thr3232 (T3232A), Ser3239 (S3239A), or both (T3232A/S3239A) were substituted with Ala were subjected to an in vitro kinase assay in the presence of [γ- 32 P]ATP. Reaction products were cleaved with thrombin to separate the Brca2 peptides from GST. Silver staining (middle panel) measures the loading of the peptides in each reaction ( ∗ ). The bottom panel shows that CDK1 catalyzes the transfer of 32 P radiolabel from [γ- 32 P]ATP to the T3232A or S3239A peptides, but not to the double-mutant T3232A/S3239A peptide. Numbers at bottom indicate relative phosphorylation normalized to the amount of peptide present in each sample.
    Figure Legend Snippet: The C-Terminal Motif of Gg Brca2 Is Functionally Cognate with Its Human Counterpart in CDK-Regulated Rad51 Binding (A) Protein sequence alignment showing the conserved cyclin-dependent kinase (CDK)-phosphorylated residues flanking Ser3239 of Gg Brca 2 and Ser3291 of Hs BRCA2. Gallus gallus residues used in this study are boxed, and residue numbers are indicated below. Asterisks indicate identical residues, double dots indicate conserved substitutions, and single dots indicate residues that are semiconserved. (B) Western blot analysis of Gg Rad51 and the myc epitope following immunoprecipitation of the myc-tagged construct encoding residues 3152–3398 of Gg Brca2 ( Gg B2 3152 aa–3398 aa ) transfected into DT40 cells. Roscovitine treatment for 30–120 min in asynchronous cells had little effect on Gg Rad51 binding (lanes 2 and 3). However, roscovitine effectively reversed the reduction in binding induced by nocodazole (compare lanes 5 and 6 with lane 4). (C and D) Western blot analysis of Gg Rad51 and the myc epitope following immunoprecipitation comparing the wild-type (WT) with S3239A/E or P3240L (C) or T3232A (D) variants of Gg B2 3152 aa–3398 aa transfected into DT40 cells. The S3239A/E and P3240L mutations in the conserved Ser-Pro consensus for CDK phosphorylation abrogated binding to Gg Rad51 in asynchronous and nocodazole-arrested mitotic cell extracts, whereas the T3232A mutation caused enhanced binding to Gg Rad51 under the same conditions. (E) Thr3232 and Ser3239 can be phosphorylated in vitro by CDK1. A wild-type Gg Brca2 peptide fused with GST (WT, sequence at top) or mutant forms in which Thr3232 (T3232A), Ser3239 (S3239A), or both (T3232A/S3239A) were substituted with Ala were subjected to an in vitro kinase assay in the presence of [γ- 32 P]ATP. Reaction products were cleaved with thrombin to separate the Brca2 peptides from GST. Silver staining (middle panel) measures the loading of the peptides in each reaction ( ∗ ). The bottom panel shows that CDK1 catalyzes the transfer of 32 P radiolabel from [γ- 32 P]ATP to the T3232A or S3239A peptides, but not to the double-mutant T3232A/S3239A peptide. Numbers at bottom indicate relative phosphorylation normalized to the amount of peptide present in each sample.

    Techniques Used: Binding Assay, Sequencing, Western Blot, Immunoprecipitation, Construct, Transfection, Mutagenesis, In Vitro, Kinase Assay, Silver Staining

    9) Product Images from "Ser649 and Ser650 Are the Major Determinants of Protein Kinase A-Mediated Activation of Human Hormone-Sensitive Lipase against Lipid Substrates"

    Article Title: Ser649 and Ser650 Are the Major Determinants of Protein Kinase A-Mediated Activation of Human Hormone-Sensitive Lipase against Lipid Substrates

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0003756

    PKA phosphorylation of HSL variants. Purified HSL variants were dephosphorylated for 1 h with calf intestinal phosphatase and then rephosphorylated by PKA in the presence of 32 P-labeled ATP. Reactions were subjected to SDS-PAGE and the amount of incorporated phosphate was determined by scintillation counting of gel bands and calculation from standards made from the original reactions.
    Figure Legend Snippet: PKA phosphorylation of HSL variants. Purified HSL variants were dephosphorylated for 1 h with calf intestinal phosphatase and then rephosphorylated by PKA in the presence of 32 P-labeled ATP. Reactions were subjected to SDS-PAGE and the amount of incorporated phosphate was determined by scintillation counting of gel bands and calculation from standards made from the original reactions.

    Techniques Used: Purification, Labeling, SDS Page

    10) Product Images from "An investigation of the structural requirements for ATP hydrolysis and DNA cleavage by the EcoKI Type I DNA restriction and modification enzyme"

    Article Title: An investigation of the structural requirements for ATP hydrolysis and DNA cleavage by the EcoKI Type I DNA restriction and modification enzyme

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr480

    3 nM pBRsk1 plasmid DNA was digested with a substoichiometric amount of nuclease (1 nM) in the presence/absence of different amounts of R subunit. The leftmost lane shows DNA size markers as in Figure 1 . The next two lanes show uncut DNA and the partial cutting of the DNA when the ratio of nuclease to DNA was 1:3. The following lanes show the same ratio of nuclease to DNA of 1:3 but with additional R subunit being added. Ratio of nuclease to R subunit for Lanes 1–4 were 1:1, 1:3, 1:4 and 1:8, respectively. Lane 5, R subunit only. Lane 6, same as Lane 4 but no ATP. Lane 6, same as Lane 4 but no SAM.
    Figure Legend Snippet: 3 nM pBRsk1 plasmid DNA was digested with a substoichiometric amount of nuclease (1 nM) in the presence/absence of different amounts of R subunit. The leftmost lane shows DNA size markers as in Figure 1 . The next two lanes show uncut DNA and the partial cutting of the DNA when the ratio of nuclease to DNA was 1:3. The following lanes show the same ratio of nuclease to DNA of 1:3 but with additional R subunit being added. Ratio of nuclease to R subunit for Lanes 1–4 were 1:1, 1:3, 1:4 and 1:8, respectively. Lane 5, R subunit only. Lane 6, same as Lane 4 but no ATP. Lane 6, same as Lane 4 but no SAM.

    Techniques Used: Plasmid Preparation

    Gel assay to test reactivation of EcoKI by RecBCD. Lane 1, undigested pBRskI (3 nM); Lane 2, partial digest of plasmid using EcoKI (1.5 nM) showing some L DNA; Lane 3, partial digest of plasmid by 1.5 nM EcoKI as in Lane 2 but in the presence of RecBCD (10 nM) showing the removal of the L DNA; Lane 4, after 5 min digestion an additional aliquot of plasmid DNA (3 nM) was added to the partial digest from Lane 2 and incubated for a further 5 min showing no further digestion but an increase in the amount of CCC DNA as expected; Lane 5, after 5 min digestion an additional aliquot of plasmid DNA (3 nM) was added to the partial digest performed in the presence of RecBCD (Lane 3) and incubated for a further 5 min showing the increase in CCC DNA but no extra digestion; Lane 6, after 5 min an additional aliquot of ATP (2 nM), SAM (0.1 mM) and plasmid DNA (3 nM) was added to the partial digest performed in the presence of RecBCD (Lane 3) and incubated for a further 5 min showing that sufficient cofactors were present.
    Figure Legend Snippet: Gel assay to test reactivation of EcoKI by RecBCD. Lane 1, undigested pBRskI (3 nM); Lane 2, partial digest of plasmid using EcoKI (1.5 nM) showing some L DNA; Lane 3, partial digest of plasmid by 1.5 nM EcoKI as in Lane 2 but in the presence of RecBCD (10 nM) showing the removal of the L DNA; Lane 4, after 5 min digestion an additional aliquot of plasmid DNA (3 nM) was added to the partial digest from Lane 2 and incubated for a further 5 min showing no further digestion but an increase in the amount of CCC DNA as expected; Lane 5, after 5 min digestion an additional aliquot of plasmid DNA (3 nM) was added to the partial digest performed in the presence of RecBCD (Lane 3) and incubated for a further 5 min showing the increase in CCC DNA but no extra digestion; Lane 6, after 5 min an additional aliquot of ATP (2 nM), SAM (0.1 mM) and plasmid DNA (3 nM) was added to the partial digest performed in the presence of RecBCD (Lane 3) and incubated for a further 5 min showing that sufficient cofactors were present.

    Techniques Used: Plasmid Preparation, Incubation, Countercurrent Chromatography

    ATP hydrolysis activity of EcoKI on various oligonucleotides duplexes as measured using the NADH-coupled enzyme assay. The assay contained 10 nM EcoKI, 5 nM of the appropriate DNA duplex given in Table 1 , 0.1 mM SAM and 2 mM ATP.
    Figure Legend Snippet: ATP hydrolysis activity of EcoKI on various oligonucleotides duplexes as measured using the NADH-coupled enzyme assay. The assay contained 10 nM EcoKI, 5 nM of the appropriate DNA duplex given in Table 1 , 0.1 mM SAM and 2 mM ATP.

    Techniques Used: Activity Assay, Nadh Coupled Enzyme Assay

    11) Product Images from "RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling"

    Article Title: RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling

    Journal: mBio

    doi: 10.1128/mBio.00833-16

    RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.
    Figure Legend Snippet: RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.

    Techniques Used: Western Blot, Incubation, RNA Binding Assay, Protease Inhibitor

    RNA mod and RIG-I trypsin sensitivity. (A to D) Digestion of 293T cell lysate for 2 h in the presence of polyU/UC RNA with the indicated modifications or canonical nucleotides (can), at increasing polyU/UC RNA concentrations (0, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 nM), in the presence of 2 mM ATP or AMP-PNP. Data represent results of Western blotting for 55-kDa and 80-kDa fragments of RIG-I with anti-helicase antibody. (E) Digestions of 293T cell lysate performed for 1.5 h in the presence of AMP-PNP and polyU/UC RNA with the indicated modifications, at increasing concentrations (0, 33, 100, 300, and 900 nM), or mass equivalent of polyI:C.
    Figure Legend Snippet: RNA mod and RIG-I trypsin sensitivity. (A to D) Digestion of 293T cell lysate for 2 h in the presence of polyU/UC RNA with the indicated modifications or canonical nucleotides (can), at increasing polyU/UC RNA concentrations (0, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 nM), in the presence of 2 mM ATP or AMP-PNP. Data represent results of Western blotting for 55-kDa and 80-kDa fragments of RIG-I with anti-helicase antibody. (E) Digestions of 293T cell lysate performed for 1.5 h in the presence of AMP-PNP and polyU/UC RNA with the indicated modifications, at increasing concentrations (0, 33, 100, 300, and 900 nM), or mass equivalent of polyI:C.

    Techniques Used: Western Blot

    12) Product Images from "GRID-seq reveals the global RNA-chromatin interactome"

    Article Title: GRID-seq reveals the global RNA-chromatin interactome

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3968

    Characterization of the GRID-seq technology a , The design of a bivalent linker for GRID-seq. The top strand is a 5′ phosphorylated DNA sequence (black) and the bottom strand consists of both DNA and RNA bases (purple) with a biotinylated T residue (red) in the middle. Randomized bases (N) served as barcodes for filtering PCR duplicates during library amplification and both ends of the linker each carry an MmeI restriction site (grey-shaded). The linker is pre-adenylated for ligation to RNA in the absence of ATP, which prevents ligation of endogenous RNAs. b , Characterization of the linker before (left) and after (right) annealing, showing the sensitivity of the RNA-containing linker to RNase A. c , Controls by omitting RNA ligase, DNA ligase or both during GRID-seq library construction, which yielded expected ligated products of singleton tags or paired-end tags (left). After adapter ligation (middle) and PCR amplification (right), the expected products were only detected after library construction with both RNA and DNA ligases.
    Figure Legend Snippet: Characterization of the GRID-seq technology a , The design of a bivalent linker for GRID-seq. The top strand is a 5′ phosphorylated DNA sequence (black) and the bottom strand consists of both DNA and RNA bases (purple) with a biotinylated T residue (red) in the middle. Randomized bases (N) served as barcodes for filtering PCR duplicates during library amplification and both ends of the linker each carry an MmeI restriction site (grey-shaded). The linker is pre-adenylated for ligation to RNA in the absence of ATP, which prevents ligation of endogenous RNAs. b , Characterization of the linker before (left) and after (right) annealing, showing the sensitivity of the RNA-containing linker to RNase A. c , Controls by omitting RNA ligase, DNA ligase or both during GRID-seq library construction, which yielded expected ligated products of singleton tags or paired-end tags (left). After adapter ligation (middle) and PCR amplification (right), the expected products were only detected after library construction with both RNA and DNA ligases.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, Ligation

    13) Product Images from "The non-canonical poly(A) polymerase FAM46C acts as an onco-suppressor in multiple myeloma"

    Article Title: The non-canonical poly(A) polymerase FAM46C acts as an onco-suppressor in multiple myeloma

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00578-5

    FAM46C interacts with RNA and is an active RNA poly(A) polymerase in vitro and in vivo. a Recombinant FAM46C WT (lanes 8–13), but not its catalytic mutant FAM46C mut (lanes 2–7), displays poly(A) polymerase activity in vitro. Reaction products (using 32 P-labeled (A) 15 as substrate) were separated in denaturing PAGE gels and visualized by autoradiography. b SDS-PAGE analysis of recombinant FAM46C WT and its catalytic mutant FAM46C mut . c FAM46D WT is an active poly(A) polymerase in vitro and requires Mn 2+ ions for its activity. Purified protein was incubated with 32 P-labeled (A) 15 primer in the presence of ATP and divalent cations as follows: Mg 2+ (lanes 4–6), both Mg 2+ /Mn 2+ (lanes 7–9), or Mn 2+ (lanes 10–12). Control reactions were carried out without the protein (lanes 1–3). d FAM46C interacts with RNA in human cells. Autoradiography of UV cross-linked 32 P-labeled RNAs co-purified with FAM46C WT GFP from HEK293 cells stably expressing the fusion protein (lanes 3–4) and from control empty cells (lanes 1–2). Immunoprecipitated RNA-protein complexes were separated by SDS-PAGE, transferred to nitrocellulose membrane, stained with Ponceau S, and subsequently autoradiographed. The right panel shows the Ponceau S stained blot merged with autoradiogram
    Figure Legend Snippet: FAM46C interacts with RNA and is an active RNA poly(A) polymerase in vitro and in vivo. a Recombinant FAM46C WT (lanes 8–13), but not its catalytic mutant FAM46C mut (lanes 2–7), displays poly(A) polymerase activity in vitro. Reaction products (using 32 P-labeled (A) 15 as substrate) were separated in denaturing PAGE gels and visualized by autoradiography. b SDS-PAGE analysis of recombinant FAM46C WT and its catalytic mutant FAM46C mut . c FAM46D WT is an active poly(A) polymerase in vitro and requires Mn 2+ ions for its activity. Purified protein was incubated with 32 P-labeled (A) 15 primer in the presence of ATP and divalent cations as follows: Mg 2+ (lanes 4–6), both Mg 2+ /Mn 2+ (lanes 7–9), or Mn 2+ (lanes 10–12). Control reactions were carried out without the protein (lanes 1–3). d FAM46C interacts with RNA in human cells. Autoradiography of UV cross-linked 32 P-labeled RNAs co-purified with FAM46C WT GFP from HEK293 cells stably expressing the fusion protein (lanes 3–4) and from control empty cells (lanes 1–2). Immunoprecipitated RNA-protein complexes were separated by SDS-PAGE, transferred to nitrocellulose membrane, stained with Ponceau S, and subsequently autoradiographed. The right panel shows the Ponceau S stained blot merged with autoradiogram

    Techniques Used: In Vitro, In Vivo, Recombinant, Mutagenesis, Activity Assay, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, SDS Page, Purification, Incubation, Stable Transfection, Expressing, Immunoprecipitation, Staining

    14) Product Images from "GSK3? phosphorylation modulates CLASP-microtubule association and lamella microtubule attachment"

    Article Title: GSK3? phosphorylation modulates CLASP-microtubule association and lamella microtubule attachment

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200901042

    Analysis of CLASP2 phosphorylation. (A–D) Metabolic labeling of tissue culture cells with [ 32 P]-labeled phosphate. EGFP-tagged CLASP2 constructs were immunoprecipitated and analyzed by SDS-PAGE. Top panels show autoradiograph, and bottom panels show the corresponding Coomassie-stained gel as loading control. Quantification of radioactivity incorporation by densitometry is shown below the gel images. (A) In both HeLa and HaCaT cells, GSK3β inhibition with 20 µM SB216763 decreases CLASP2(340–1,084) phosphorylation. Mutation of the GSK3β motif between S594 to S614 (6×S/A) eliminates GSK3β-dependent phosphorylation. (B) Mutation of individual serine residues between S594 and S614 shows that S614 is not part of the motif and reveals hierarchical phosphorylation by GSK3β. (C) The domain required for CLASP2 association along lamella MTs (875–1,084) is not required for efficient phosphorylation by GSK3β. (D) Combined mutation of the GSK3β motifs between S568 to S576 and S594 to S614 (9×S/A) is required to completely abolish phosphorylation of the MT plus end tracking domain CLASP2(512–650) by constitutively active GSK3β(S9A). (E) In vitro phosphorylation of immunoprecipitated EGFP-CLASP2(512–650) by purified GSK3β in the presence of γ-[ 32 P]ATP.
    Figure Legend Snippet: Analysis of CLASP2 phosphorylation. (A–D) Metabolic labeling of tissue culture cells with [ 32 P]-labeled phosphate. EGFP-tagged CLASP2 constructs were immunoprecipitated and analyzed by SDS-PAGE. Top panels show autoradiograph, and bottom panels show the corresponding Coomassie-stained gel as loading control. Quantification of radioactivity incorporation by densitometry is shown below the gel images. (A) In both HeLa and HaCaT cells, GSK3β inhibition with 20 µM SB216763 decreases CLASP2(340–1,084) phosphorylation. Mutation of the GSK3β motif between S594 to S614 (6×S/A) eliminates GSK3β-dependent phosphorylation. (B) Mutation of individual serine residues between S594 and S614 shows that S614 is not part of the motif and reveals hierarchical phosphorylation by GSK3β. (C) The domain required for CLASP2 association along lamella MTs (875–1,084) is not required for efficient phosphorylation by GSK3β. (D) Combined mutation of the GSK3β motifs between S568 to S576 and S594 to S614 (9×S/A) is required to completely abolish phosphorylation of the MT plus end tracking domain CLASP2(512–650) by constitutively active GSK3β(S9A). (E) In vitro phosphorylation of immunoprecipitated EGFP-CLASP2(512–650) by purified GSK3β in the presence of γ-[ 32 P]ATP.

    Techniques Used: Labeling, Construct, Immunoprecipitation, SDS Page, Autoradiography, Staining, Radioactivity, Inhibition, Mutagenesis, In Vitro, Purification

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    Transmigration Assay:

    Article Title: Release of monocyte migration signals by breast cancer cell lines after ablative and fractionated ?-irradiation
    Article Snippet: Apyrase treatment was carried out by adding 500 milliunits of nucleotide diphosphohydrolase (apyrase, New England Biolabs) to 1.5 ml culture supernatant and allowing nucleotide degradation for 30–50 min at 37°C. .. Apyrase treatment was carried out by adding 500 milliunits of nucleotide diphosphohydrolase (apyrase, New England Biolabs) to 1.5 ml culture supernatant and allowing nucleotide degradation for 30–50 min at 37°C.

    Transwell Migration Assay:

    Article Title: Release of monocyte migration signals by breast cancer cell lines after ablative and fractionated ?-irradiation
    Article Snippet: Paragraph title: Transwell migration assay ... Apyrase treatment was carried out by adding 500 milliunits of nucleotide diphosphohydrolase (apyrase, New England Biolabs) to 1.5 ml culture supernatant and allowing nucleotide degradation for 30–50 min at 37°C.

    Western Blot:

    Article Title: SAF-A Regulates Interphase Chromosome Structure through Oligomerization with Chromatin-Associated RNAs
    Article Snippet: After quenching DSS with 20 mM Tris pH 7.0, BM(PEG)2 with 20 mM L-cysteine (Sigma-Aldrich) or GMBS with 20 mM Tris pH 7.0 and 20 mM L-cysteine, cells were processed for western blotting. .. For D, after pre-treatment with 3 μg.ml-1 PureLink RNaseA (ThermoFisher) in reaction buffer at 37°C for 10 min, 293T cell lysates were incubated in the presence or absence of 30 μg.ml-1 total RNA from 293T cells and 5 units.ml-1 Apyrase (NEB) at 37°C for 10 min, following by crosslinking.

    Hybridization:

    Article Title: Ku Heterodimer-Independent End Joining in Trypanosoma brucei Cell Extracts Relies upon Sequence Microhomology
    Article Snippet: To deplete ATP, 10 U of apyrase (New England Biolabs) was added to each reaction mixture, and the extract was incubated for 10 min at 37°C prior to substrate addition. .. To deplete ATP, 10 U of apyrase (New England Biolabs) was added to each reaction mixture, and the extract was incubated for 10 min at 37°C prior to substrate addition.

    Countercurrent Chromatography:

    Article Title: Release of monocyte migration signals by breast cancer cell lines after ablative and fractionated ?-irradiation
    Article Snippet: Full length PCR products were generated from cDNA (80 ng per reaction) by employing 5 units HotStar HiFidelity DNA-Polymerase in 1 × HotStar HiFidelity reaction buffer, and 1 × Q solution (all from Qiagen, Hilden, Germany) in the presence of 1 μM of each primer (p53 Forward 5′-ATG GAG GAG CCG CAG TCA G-3′, p53 Reverse 5′-TCA GTC TGA GTC AGG CCC TTC T-3′, synthesized by Sigma-Aldrich, Taufkirchen Germany) in 100 μl final volume (cycling program: 1 × 5′ 95°C; 40 × 15″ 95°C, 1′ 60°C, 1′30″ 72°C; 1 × 10′ 72°C). .. The chemokines SDF-1α and WKYMVm (agonist of formyl-peptide receptors 1, 2, and 3) were from R & D Systems, adenosine 5′-triphosphate disodium salt (ATP) from Sigma-Aldrich, and nucleotide diphosphohydrolase (apyrase) was purchased from New England Biolabs (Frankfurt, Germany).

    Transfection:

    Article Title: Nanodelivery of a functional membrane receptor to manipulate cellular phenotype
    Article Snippet: For the β2 AR functionality test, cells were plated onto 100 mm petri dishes (VWR) at a density of 200,000 per dish and grown for 24 h before being transfected with 3 μg of Gs-mCherry plasmid DNA. .. Cell lysates were prepared by cell titration in apyrase reaction buffer (NEB) supplemented with protease inhibitors cocktail (Sigma) (20 mM MES, 50 mM NaCl, 5 mM CaCl2 , 1 mM DTT, 0.05% Tween-20, pH 6.5).

    Generated:

    Article Title: Release of monocyte migration signals by breast cancer cell lines after ablative and fractionated ?-irradiation
    Article Snippet: Full length PCR products were generated from cDNA (80 ng per reaction) by employing 5 units HotStar HiFidelity DNA-Polymerase in 1 × HotStar HiFidelity reaction buffer, and 1 × Q solution (all from Qiagen, Hilden, Germany) in the presence of 1 μM of each primer (p53 Forward 5′-ATG GAG GAG CCG CAG TCA G-3′, p53 Reverse 5′-TCA GTC TGA GTC AGG CCC TTC T-3′, synthesized by Sigma-Aldrich, Taufkirchen Germany) in 100 μl final volume (cycling program: 1 × 5′ 95°C; 40 × 15″ 95°C, 1′ 60°C, 1′30″ 72°C; 1 × 10′ 72°C). .. The chemokines SDF-1α and WKYMVm (agonist of formyl-peptide receptors 1, 2, and 3) were from R & D Systems, adenosine 5′-triphosphate disodium salt (ATP) from Sigma-Aldrich, and nucleotide diphosphohydrolase (apyrase) was purchased from New England Biolabs (Frankfurt, Germany).

    Article Title: Mechanism of Parkin activation by PINK1
    Article Snippet: The membrane was then washed with PBS-T, incubated for 1 h at room temperature with anti-mouse IgG-HRP (NXA931, GE Healthcare) in 5% (w/v) milk in PBS-T, washed in PBS-T and visualised using the Amersham Western Blotting Detection Reagent (GE Healthcare) and a ChemiDoc Touch Imaging System (BioRad). .. The UBE2D3~Ub conjugate was generated by incubating UBE2D3 (20 μM) with Hs UBE1 (20 nM) and Ub (80 μM) in ubiquitination buffer supplemented with 5 μM CaCl2 at 37°C for 10 min. To remove remaining ATP, 0.5 U of Apyrase (NEB) were added and the reaction incubated at 30°C for 30 min. .. The discharge reaction was studied by addition of 1 μM phospho-Parkin wild-type or R104A to a diluted charging reaction mixture (final UBE2D3 concentration was 9 μM).

    Imaging:

    Article Title: RNF12 X-Linked Intellectual Disability Mutations Disrupt E3 Ligase Activity and Neural Differentiation
    Article Snippet: Reaction was stopped by depleting ATP with 4.5 U/mL apyrase (New England Biolabs) for 10 min at RT. .. Reactions were stopped with nonreducing SDS loading buffer and analyzed via SDS-PAGE.

    Sequencing:

    Article Title: Release of monocyte migration signals by breast cancer cell lines after ablative and fractionated ?-irradiation
    Article Snippet: The amplicons were purified by the NucleoSpin Extract II Kit (Macherey & Nagel, Dueren, Germany), and sequencing was performed by Seqlab Sequencing Services (Goettingen, Germany). .. The chemokines SDF-1α and WKYMVm (agonist of formyl-peptide receptors 1, 2, and 3) were from R & D Systems, adenosine 5′-triphosphate disodium salt (ATP) from Sigma-Aldrich, and nucleotide diphosphohydrolase (apyrase) was purchased from New England Biolabs (Frankfurt, Germany).

    Article Title: Reversible methylation of m6Am in the 5′ cap controls mRNA stability
    Article Snippet: Thus, this method quantifies m6 A in a GA sequence context. .. 5′ ends were subsequently labelled with 10 units of T4 PNK (NEB) and 0.4 mBq [γ-32 P] ATP at 37 °C for 30 min followed by removal of the γ-phosphate of ATP by incubation with 10 units Apyrase (NEB) at 30 °C for 30 min. After phenol-chloroform extraction and ethanol precipitation, RNA samples were resuspended in 10 μl of DEPC-H2 O and digested to single nucleotides with 2 units of P1 nuclease (Sigma) for 3 h at 37 °C.

    Sonication:

    Article Title: Concomitant activation of P2Y2 and P2Y6 receptors on monocytes is required for TLR1/2-induced neutrophil migration by regulating IL-8 secretion
    Article Snippet: Recombinant and semi-purified potato apyrase grade VII were bought from New England BioLabs (Ontario, Canada) and Sigma, respectively. .. Recombinant and semi-purified potato apyrase grade VII were bought from New England BioLabs (Ontario, Canada) and Sigma, respectively.

    Recombinant:

    Article Title: Concomitant activation of P2Y2 and P2Y6 receptors on monocytes is required for TLR1/2-induced neutrophil migration by regulating IL-8 secretion
    Article Snippet: MRS2500, MRS2578, NF157, KN62 and A438079 were purchased from Tocris Bioscience (Bristol, UK) and RB2 from ICN Biochemicals, (Aurora, OH, USA). .. Recombinant and semi-purified potato apyrase grade VII were bought from New England BioLabs (Ontario, Canada) and Sigma, respectively. .. Human recombinant IL-8 (hrIL-8) was purchased from Medicorp (Montreal, Canada), IL-8 neutralizing Ab MAB208 from R & D Systems (Minneapolis, MN, USA) and a matching isotype mouse IgG1 Ab (used as a control in Boyden chamber transmigration assays) from Sigma.

    Pull Down Assay:

    Article Title: Nanodelivery of a functional membrane receptor to manipulate cellular phenotype
    Article Snippet: Paragraph title: Single-molecule pull-down assay (SiMPull) ... Cell lysates were prepared by cell titration in apyrase reaction buffer (NEB) supplemented with protease inhibitors cocktail (Sigma) (20 mM MES, 50 mM NaCl, 5 mM CaCl2 , 1 mM DTT, 0.05% Tween-20, pH 6.5).

    Fluorescence:

    Article Title: Release of monocyte migration signals by breast cancer cell lines after ablative and fractionated ?-irradiation
    Article Snippet: Green calcein fluorescence was quantified with a Synergy MX fluorescence reader (BioTek Instrumtents GmbH, Bad Friedrichshall, Germany), and transmigration was calculated as percentage of total cells deployed. .. Apyrase treatment was carried out by adding 500 milliunits of nucleotide diphosphohydrolase (apyrase, New England Biolabs) to 1.5 ml culture supernatant and allowing nucleotide degradation for 30–50 min at 37°C.

    Cell Stimulation:

    Article Title: Concomitant activation of P2Y2 and P2Y6 receptors on monocytes is required for TLR1/2-induced neutrophil migration by regulating IL-8 secretion
    Article Snippet: Recombinant and semi-purified potato apyrase grade VII were bought from New England BioLabs (Ontario, Canada) and Sigma, respectively. .. Recombinant and semi-purified potato apyrase grade VII were bought from New England BioLabs (Ontario, Canada) and Sigma, respectively.

    Labeling:

    Article Title: The N6-methyladenosine (m6A)-forming enzyme METTL3 controls myeloid differentiation of normal and leukemia cells
    Article Snippet: 100 ng of poly(A) purified RNA was digested with 2U ribonuclease T1 for 2h at 37°C in the presence of RNAseOUT (Invitrogen). .. Digested RNA was 5′ labeled with 0.4 mBq [γ-32 P] ATP for 30 min at 37°C followed by removal of the γ– phosphate of ATP by incubation with 10U apyrase (NEB) for 30 min at 30°C. .. RNA was purified by phenol-chloroform extraction and ethanol precipitation and resuspended in 10 μl of DEPC-H2 O and digested to single nucleotides with 2U of P1 nuclease for 3h at 37°C.

    Titration:

    Article Title: Nanodelivery of a functional membrane receptor to manipulate cellular phenotype
    Article Snippet: For the β2 AR functionality test, cells were plated onto 100 mm petri dishes (VWR) at a density of 200,000 per dish and grown for 24 h before being transfected with 3 μg of Gs-mCherry plasmid DNA. .. Cell lysates were prepared by cell titration in apyrase reaction buffer (NEB) supplemented with protease inhibitors cocktail (Sigma) (20 mM MES, 50 mM NaCl, 5 mM CaCl2 , 1 mM DTT, 0.05% Tween-20, pH 6.5). .. Complete cell lysis was ensured by probe-sonicating on ice for 30 s. Cell debris was removed by 10 min of centrifugation at 14,000 rcf (4 °C).

    Polymerase Chain Reaction:

    Article Title: Release of monocyte migration signals by breast cancer cell lines after ablative and fractionated ?-irradiation
    Article Snippet: Full length PCR products were generated from cDNA (80 ng per reaction) by employing 5 units HotStar HiFidelity DNA-Polymerase in 1 × HotStar HiFidelity reaction buffer, and 1 × Q solution (all from Qiagen, Hilden, Germany) in the presence of 1 μM of each primer (p53 Forward 5′-ATG GAG GAG CCG CAG TCA G-3′, p53 Reverse 5′-TCA GTC TGA GTC AGG CCC TTC T-3′, synthesized by Sigma-Aldrich, Taufkirchen Germany) in 100 μl final volume (cycling program: 1 × 5′ 95°C; 40 × 15″ 95°C, 1′ 60°C, 1′30″ 72°C; 1 × 10′ 72°C). .. The chemokines SDF-1α and WKYMVm (agonist of formyl-peptide receptors 1, 2, and 3) were from R & D Systems, adenosine 5′-triphosphate disodium salt (ATP) from Sigma-Aldrich, and nucleotide diphosphohydrolase (apyrase) was purchased from New England Biolabs (Frankfurt, Germany).

    Article Title: Ku Heterodimer-Independent End Joining in Trypanosoma brucei Cell Extracts Relies upon Sequence Microhomology
    Article Snippet: To deplete ATP, 10 U of apyrase (New England Biolabs) was added to each reaction mixture, and the extract was incubated for 10 min at 37°C prior to substrate addition. .. To deplete ATP, 10 U of apyrase (New England Biolabs) was added to each reaction mixture, and the extract was incubated for 10 min at 37°C prior to substrate addition.

    Lysis:

    Article Title: Release of monocyte migration signals by breast cancer cell lines after ablative and fractionated ?-irradiation
    Article Snippet: Subsequently, the cells in the lower chamber were collected by centrifugation and lysed in 100 μl lysis buffer (20 mM HEPES-K pH 7.4, 84 mM KCl, 10 mM MgCl2 , 0.2 mM EDTA, 0.2 mM EGTA, 0.5% Igepal). .. Apyrase treatment was carried out by adding 500 milliunits of nucleotide diphosphohydrolase (apyrase, New England Biolabs) to 1.5 ml culture supernatant and allowing nucleotide degradation for 30–50 min at 37°C.

    Purification:

    Article Title: Release of monocyte migration signals by breast cancer cell lines after ablative and fractionated ?-irradiation
    Article Snippet: The amplicons were purified by the NucleoSpin Extract II Kit (Macherey & Nagel, Dueren, Germany), and sequencing was performed by Seqlab Sequencing Services (Goettingen, Germany). .. The chemokines SDF-1α and WKYMVm (agonist of formyl-peptide receptors 1, 2, and 3) were from R & D Systems, adenosine 5′-triphosphate disodium salt (ATP) from Sigma-Aldrich, and nucleotide diphosphohydrolase (apyrase) was purchased from New England Biolabs (Frankfurt, Germany).

    Article Title: The N6-methyladenosine (m6A)-forming enzyme METTL3 controls myeloid differentiation of normal and leukemia cells
    Article Snippet: 100 ng of poly(A) purified RNA was digested with 2U ribonuclease T1 for 2h at 37°C in the presence of RNAseOUT (Invitrogen). .. Digested RNA was 5′ labeled with 0.4 mBq [γ-32 P] ATP for 30 min at 37°C followed by removal of the γ– phosphate of ATP by incubation with 10U apyrase (NEB) for 30 min at 30°C.

    Article Title: Ku Heterodimer-Independent End Joining in Trypanosoma brucei Cell Extracts Relies upon Sequence Microhomology
    Article Snippet: To deplete ATP, 10 U of apyrase (New England Biolabs) was added to each reaction mixture, and the extract was incubated for 10 min at 37°C prior to substrate addition. .. To deplete ATP, 10 U of apyrase (New England Biolabs) was added to each reaction mixture, and the extract was incubated for 10 min at 37°C prior to substrate addition.

    SDS Page:

    Article Title: RNF12 X-Linked Intellectual Disability Mutations Disrupt E3 Ligase Activity and Neural Differentiation
    Article Snippet: Reaction was stopped by depleting ATP with 4.5 U/mL apyrase (New England Biolabs) for 10 min at RT. .. Then, 40 μM UBE2D1-ubiquitin was incubated with 280 nM RNF12 and 150 mM L-lysine in a buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5 mM TCEP, and 0.1% (v/v) NP-40 at RT.

    Plasmid Preparation:

    Article Title: Nanodelivery of a functional membrane receptor to manipulate cellular phenotype
    Article Snippet: For the β2 AR functionality test, cells were plated onto 100 mm petri dishes (VWR) at a density of 200,000 per dish and grown for 24 h before being transfected with 3 μg of Gs-mCherry plasmid DNA. .. Cell lysates were prepared by cell titration in apyrase reaction buffer (NEB) supplemented with protease inhibitors cocktail (Sigma) (20 mM MES, 50 mM NaCl, 5 mM CaCl2 , 1 mM DTT, 0.05% Tween-20, pH 6.5).

    Software:

    Article Title: RNF12 X-Linked Intellectual Disability Mutations Disrupt E3 Ligase Activity and Neural Differentiation
    Article Snippet: Reaction was stopped by depleting ATP with 4.5 U/mL apyrase (New England Biolabs) for 10 min at RT. .. Reactions were stopped with nonreducing SDS loading buffer and analyzed via SDS-PAGE.

    Agarose Gel Electrophoresis:

    Article Title: Ku Heterodimer-Independent End Joining in Trypanosoma brucei Cell Extracts Relies upon Sequence Microhomology
    Article Snippet: To deplete ATP, 10 U of apyrase (New England Biolabs) was added to each reaction mixture, and the extract was incubated for 10 min at 37°C prior to substrate addition. .. To deplete ATP, 10 U of apyrase (New England Biolabs) was added to each reaction mixture, and the extract was incubated for 10 min at 37°C prior to substrate addition.

    Ethanol Precipitation:

    Article Title: Ku Heterodimer-Independent End Joining in Trypanosoma brucei Cell Extracts Relies upon Sequence Microhomology
    Article Snippet: To deplete ATP, 10 U of apyrase (New England Biolabs) was added to each reaction mixture, and the extract was incubated for 10 min at 37°C prior to substrate addition. .. To deplete ATP, 10 U of apyrase (New England Biolabs) was added to each reaction mixture, and the extract was incubated for 10 min at 37°C prior to substrate addition.

    Article Title: m6A RNA methylation promotes XIST-mediated transcriptional repression
    Article Snippet: In brief, poly(A) RNA (100 ng) was digested with 2 U RNase T1 (Thermo Fisher) for 2 h at 37 °C in the presence of RNasin RNase Inhibitor (Promega). .. Five prime ends were subsequently labelled with 10 U T4 PNK (NEB) and 0.4 mBq [γ-32 P]ATP at 37 °C for 30 min followed by removal of the γ-phosphate of ATP by incubation with 10 U apyrase (NEB) at 30 °C for 30 min. After phenol-chloroform extraction and ethanol precipitation, RNA samples were resuspended in 10 μl of water and digested to mononucleotides with 2 U of P1 nuclease (Sigma-Aldrich) for 3 h at 37 °C. .. Following this, 2 μl of the released 5′ monophosphates from this digest were then analysed by 2D-TLC on glass-backed PEI-cellulose plates (Merck-Millipore).

    Article Title: Reversible methylation of m6Am in the 5′ cap controls mRNA stability
    Article Snippet: Thus, this method quantifies m6 A in a GA sequence context. .. 5′ ends were subsequently labelled with 10 units of T4 PNK (NEB) and 0.4 mBq [γ-32 P] ATP at 37 °C for 30 min followed by removal of the γ-phosphate of ATP by incubation with 10 units Apyrase (NEB) at 30 °C for 30 min. After phenol-chloroform extraction and ethanol precipitation, RNA samples were resuspended in 10 μl of DEPC-H2 O and digested to single nucleotides with 2 units of P1 nuclease (Sigma) for 3 h at 37 °C. .. 1 μl of the released 5′ monophosphates from this digest were then analysed by 2D TLC on glass-backed PEI-cellulose plates (MerckMillipore) as described previously .

    Concentration Assay:

    Article Title: Ku Heterodimer-Independent End Joining in Trypanosoma brucei Cell Extracts Relies upon Sequence Microhomology
    Article Snippet: To deplete ATP, 10 U of apyrase (New England Biolabs) was added to each reaction mixture, and the extract was incubated for 10 min at 37°C prior to substrate addition. .. To deplete ATP, 10 U of apyrase (New England Biolabs) was added to each reaction mixture, and the extract was incubated for 10 min at 37°C prior to substrate addition.

    Article Title: RNF12 X-Linked Intellectual Disability Mutations Disrupt E3 Ligase Activity and Neural Differentiation
    Article Snippet: Reaction was stopped by depleting ATP with 4.5 U/mL apyrase (New England Biolabs) for 10 min at RT. .. Gels were stained with Coomassie staining (InstantBlue reagent, Expedeon) and scanned in an Odyssey CLx Infrared Imaging System (LI-COR Biosciences), and the protein signal was quantified using Image Studio software (LI-COR Biosciences).

    Thin Layer Chromatography:

    Article Title: The N6-methyladenosine (m6A)-forming enzyme METTL3 controls myeloid differentiation of normal and leukemia cells
    Article Snippet: Paragraph title: Determination of relative m6 A levels by two-dimensional thin layer chromatography ... Digested RNA was 5′ labeled with 0.4 mBq [γ-32 P] ATP for 30 min at 37°C followed by removal of the γ– phosphate of ATP by incubation with 10U apyrase (NEB) for 30 min at 30°C.

    Article Title: m6A RNA methylation promotes XIST-mediated transcriptional repression
    Article Snippet: Paragraph title: Determination of relative m6 A levels by thin layer chromatography ... Five prime ends were subsequently labelled with 10 U T4 PNK (NEB) and 0.4 mBq [γ-32 P]ATP at 37 °C for 30 min followed by removal of the γ-phosphate of ATP by incubation with 10 U apyrase (NEB) at 30 °C for 30 min. After phenol-chloroform extraction and ethanol precipitation, RNA samples were resuspended in 10 μl of water and digested to mononucleotides with 2 U of P1 nuclease (Sigma-Aldrich) for 3 h at 37 °C.

    Article Title: Reversible methylation of m6Am in the 5′ cap controls mRNA stability
    Article Snippet: Paragraph title: Determination of relative m6 Am , Am and m6 A levels by thin layer chromatography ... 5′ ends were subsequently labelled with 10 units of T4 PNK (NEB) and 0.4 mBq [γ-32 P] ATP at 37 °C for 30 min followed by removal of the γ-phosphate of ATP by incubation with 10 units Apyrase (NEB) at 30 °C for 30 min. After phenol-chloroform extraction and ethanol precipitation, RNA samples were resuspended in 10 μl of DEPC-H2 O and digested to single nucleotides with 2 units of P1 nuclease (Sigma) for 3 h at 37 °C.

    Chemotaxis Assay:

    Article Title: Release of monocyte migration signals by breast cancer cell lines after ablative and fractionated ?-irradiation
    Article Snippet: Apyrase treatment was carried out by adding 500 milliunits of nucleotide diphosphohydrolase (apyrase, New England Biolabs) to 1.5 ml culture supernatant and allowing nucleotide degradation for 30–50 min at 37°C. .. Apyrase treatment was carried out by adding 500 milliunits of nucleotide diphosphohydrolase (apyrase, New England Biolabs) to 1.5 ml culture supernatant and allowing nucleotide degradation for 30–50 min at 37°C.

    Migration:

    Article Title: Release of monocyte migration signals by breast cancer cell lines after ablative and fractionated ?-irradiation
    Article Snippet: Apyrase treatment was carried out by adding 500 milliunits of nucleotide diphosphohydrolase (apyrase, New England Biolabs) to 1.5 ml culture supernatant and allowing nucleotide degradation for 30–50 min at 37°C. .. Apyrase treatment was carried out by adding 500 milliunits of nucleotide diphosphohydrolase (apyrase, New England Biolabs) to 1.5 ml culture supernatant and allowing nucleotide degradation for 30–50 min at 37°C.

    Staining:

    Article Title: Mechanism of Parkin activation by PINK1
    Article Snippet: The UBE2D3~Ub conjugate was generated by incubating UBE2D3 (20 μM) with Hs UBE1 (20 nM) and Ub (80 μM) in ubiquitination buffer supplemented with 5 μM CaCl2 at 37°C for 10 min. To remove remaining ATP, 0.5 U of Apyrase (NEB) were added and the reaction incubated at 30°C for 30 min. .. The UBE2D3~Ub conjugate was generated by incubating UBE2D3 (20 μM) with Hs UBE1 (20 nM) and Ub (80 μM) in ubiquitination buffer supplemented with 5 μM CaCl2 at 37°C for 10 min. To remove remaining ATP, 0.5 U of Apyrase (NEB) were added and the reaction incubated at 30°C for 30 min.

    Article Title: RNF12 X-Linked Intellectual Disability Mutations Disrupt E3 Ligase Activity and Neural Differentiation
    Article Snippet: Reaction was stopped by depleting ATP with 4.5 U/mL apyrase (New England Biolabs) for 10 min at RT. .. Reactions were stopped with nonreducing SDS loading buffer and analyzed via SDS-PAGE.

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    New England Biolabs m atp
    ER Ca 2+ release mediated the effect of <t>ATP</t> on <t>eNOS</t> Ser-635 phosphorylation in BAECs. A , effects of BAPTA-AM (50 μ m ) on ATP-induced ERK1/2 activation and eNOS Ser-635 phosphorylation. BAECs were preloaded with BAPTA-AM for 30 min and then exposed
    M Atp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m atp/product/New England Biolabs
    Average 89 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    m atp - by Bioz Stars, 2019-12
    89/100 stars
      Buy from Supplier

    99
    New England Biolabs atp
    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM <t>ATP</t> and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM <t>DTT,</t> 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Atp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atp/product/New England Biolabs
    Average 99 stars, based on 0 article reviews
    Price from $9.99 to $1999.99
    atp - by Bioz Stars, 2019-12
    99/100 stars
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    Image Search Results


    ER Ca 2+ release mediated the effect of ATP on eNOS Ser-635 phosphorylation in BAECs. A , effects of BAPTA-AM (50 μ m ) on ATP-induced ERK1/2 activation and eNOS Ser-635 phosphorylation. BAECs were preloaded with BAPTA-AM for 30 min and then exposed

    Journal:

    Article Title: Endoplasmic Reticulum Ca2

    doi: 10.1074/jbc.M111.220236

    Figure Lengend Snippet: ER Ca 2+ release mediated the effect of ATP on eNOS Ser-635 phosphorylation in BAECs. A , effects of BAPTA-AM (50 μ m ) on ATP-induced ERK1/2 activation and eNOS Ser-635 phosphorylation. BAECs were preloaded with BAPTA-AM for 30 min and then exposed

    Article Snippet: In vitro phosphorylation was performed in a 50-μl reaction system containing 50 m m Tris-HCl, pH 7.4, 10 m m MgCl2 , 2 m m DTT, 1 m m EGTA, 0.01% Brij 35, 0.5 m m ATP, eNOS (0.1 μg), and ERK2 (100 units; New England Biolabs, Beverly, MA).

    Techniques: Activation Assay

    Mutation of Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. A , effects of ATP (20 μ m ) on eNOS Ser-635 phosphorylation in WT eNOS and S635A eNOS-transfect cells. Representative blots are shown from three independent

    Journal:

    Article Title: Endoplasmic Reticulum Ca2

    doi: 10.1074/jbc.M111.220236

    Figure Lengend Snippet: Mutation of Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. A , effects of ATP (20 μ m ) on eNOS Ser-635 phosphorylation in WT eNOS and S635A eNOS-transfect cells. Representative blots are shown from three independent

    Article Snippet: In vitro phosphorylation was performed in a 50-μl reaction system containing 50 m m Tris-HCl, pH 7.4, 10 m m MgCl2 , 2 m m DTT, 1 m m EGTA, 0.01% Brij 35, 0.5 m m ATP, eNOS (0.1 μg), and ERK2 (100 units; New England Biolabs, Beverly, MA).

    Techniques: Mutagenesis

    ATP triggered eNOS Ser-635 phosphorylation in BAECs. A , time course effects of ATP (20 μ m ) on eNOS phosphorylation. Representative blots are shown from five independent experiments. B , quantitative analyses of the effects of ATP on eNOS Ser-1179,

    Journal:

    Article Title: Endoplasmic Reticulum Ca2

    doi: 10.1074/jbc.M111.220236

    Figure Lengend Snippet: ATP triggered eNOS Ser-635 phosphorylation in BAECs. A , time course effects of ATP (20 μ m ) on eNOS phosphorylation. Representative blots are shown from five independent experiments. B , quantitative analyses of the effects of ATP on eNOS Ser-1179,

    Article Snippet: In vitro phosphorylation was performed in a 50-μl reaction system containing 50 m m Tris-HCl, pH 7.4, 10 m m MgCl2 , 2 m m DTT, 1 m m EGTA, 0.01% Brij 35, 0.5 m m ATP, eNOS (0.1 μg), and ERK2 (100 units; New England Biolabs, Beverly, MA).

    Techniques:

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Journal: PLoS ONE

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    doi: 10.1371/journal.pone.0190062

    Figure Lengend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Article Snippet: T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) as a 10x stock, NEBNext® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) as a 5x stock, restriction endonucleases EcoRV, NruI, BstNI, Hpy188I, NdeI, BamHI, as well as λ DNA, 1 M DTT, 6x Purple Loading Dye with SDS, Proteinase K and 10 mM ATP were obtained from New England Biolabs (NEB, Ipswich, MA).

    Techniques: Ligation, Agarose Gel Electrophoresis, Staining

    Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Journal: PLoS ONE

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    doi: 10.1371/journal.pone.0190062

    Figure Lengend Snippet: Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Article Snippet: T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) as a 10x stock, NEBNext® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) as a 5x stock, restriction endonucleases EcoRV, NruI, BstNI, Hpy188I, NdeI, BamHI, as well as λ DNA, 1 M DTT, 6x Purple Loading Dye with SDS, Proteinase K and 10 mM ATP were obtained from New England Biolabs (NEB, Ipswich, MA).

    Techniques: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Journal: PLoS ONE

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    doi: 10.1371/journal.pone.0190062

    Figure Lengend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Article Snippet: T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) as a 10x stock, NEBNext® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) as a 5x stock, restriction endonucleases EcoRV, NruI, BstNI, Hpy188I, NdeI, BamHI, as well as λ DNA, 1 M DTT, 6x Purple Loading Dye with SDS, Proteinase K and 10 mM ATP were obtained from New England Biolabs (NEB, Ipswich, MA).

    Techniques: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Journal: PLoS ONE

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    doi: 10.1371/journal.pone.0190062

    Figure Lengend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Article Snippet: T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) as a 10x stock, NEBNext® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) as a 5x stock, restriction endonucleases EcoRV, NruI, BstNI, Hpy188I, NdeI, BamHI, as well as λ DNA, 1 M DTT, 6x Purple Loading Dye with SDS, Proteinase K and 10 mM ATP were obtained from New England Biolabs (NEB, Ipswich, MA).

    Techniques: Ligation, Produced, Standard Deviation

    3 nM pBRsk1 plasmid DNA was digested with a substoichiometric amount of nuclease (1 nM) in the presence/absence of different amounts of R subunit. The leftmost lane shows DNA size markers as in Figure 1 . The next two lanes show uncut DNA and the partial cutting of the DNA when the ratio of nuclease to DNA was 1:3. The following lanes show the same ratio of nuclease to DNA of 1:3 but with additional R subunit being added. Ratio of nuclease to R subunit for Lanes 1–4 were 1:1, 1:3, 1:4 and 1:8, respectively. Lane 5, R subunit only. Lane 6, same as Lane 4 but no ATP. Lane 6, same as Lane 4 but no SAM.

    Journal: Nucleic Acids Research

    Article Title: An investigation of the structural requirements for ATP hydrolysis and DNA cleavage by the EcoKI Type I DNA restriction and modification enzyme

    doi: 10.1093/nar/gkr480

    Figure Lengend Snippet: 3 nM pBRsk1 plasmid DNA was digested with a substoichiometric amount of nuclease (1 nM) in the presence/absence of different amounts of R subunit. The leftmost lane shows DNA size markers as in Figure 1 . The next two lanes show uncut DNA and the partial cutting of the DNA when the ratio of nuclease to DNA was 1:3. The following lanes show the same ratio of nuclease to DNA of 1:3 but with additional R subunit being added. Ratio of nuclease to R subunit for Lanes 1–4 were 1:1, 1:3, 1:4 and 1:8, respectively. Lane 5, R subunit only. Lane 6, same as Lane 4 but no ATP. Lane 6, same as Lane 4 but no SAM.

    Article Snippet: ATP, SAM and EcoRI were purchased from New England Biolabs.

    Techniques: Plasmid Preparation

    Gel assay to test reactivation of EcoKI by RecBCD. Lane 1, undigested pBRskI (3 nM); Lane 2, partial digest of plasmid using EcoKI (1.5 nM) showing some L DNA; Lane 3, partial digest of plasmid by 1.5 nM EcoKI as in Lane 2 but in the presence of RecBCD (10 nM) showing the removal of the L DNA; Lane 4, after 5 min digestion an additional aliquot of plasmid DNA (3 nM) was added to the partial digest from Lane 2 and incubated for a further 5 min showing no further digestion but an increase in the amount of CCC DNA as expected; Lane 5, after 5 min digestion an additional aliquot of plasmid DNA (3 nM) was added to the partial digest performed in the presence of RecBCD (Lane 3) and incubated for a further 5 min showing the increase in CCC DNA but no extra digestion; Lane 6, after 5 min an additional aliquot of ATP (2 nM), SAM (0.1 mM) and plasmid DNA (3 nM) was added to the partial digest performed in the presence of RecBCD (Lane 3) and incubated for a further 5 min showing that sufficient cofactors were present.

    Journal: Nucleic Acids Research

    Article Title: An investigation of the structural requirements for ATP hydrolysis and DNA cleavage by the EcoKI Type I DNA restriction and modification enzyme

    doi: 10.1093/nar/gkr480

    Figure Lengend Snippet: Gel assay to test reactivation of EcoKI by RecBCD. Lane 1, undigested pBRskI (3 nM); Lane 2, partial digest of plasmid using EcoKI (1.5 nM) showing some L DNA; Lane 3, partial digest of plasmid by 1.5 nM EcoKI as in Lane 2 but in the presence of RecBCD (10 nM) showing the removal of the L DNA; Lane 4, after 5 min digestion an additional aliquot of plasmid DNA (3 nM) was added to the partial digest from Lane 2 and incubated for a further 5 min showing no further digestion but an increase in the amount of CCC DNA as expected; Lane 5, after 5 min digestion an additional aliquot of plasmid DNA (3 nM) was added to the partial digest performed in the presence of RecBCD (Lane 3) and incubated for a further 5 min showing the increase in CCC DNA but no extra digestion; Lane 6, after 5 min an additional aliquot of ATP (2 nM), SAM (0.1 mM) and plasmid DNA (3 nM) was added to the partial digest performed in the presence of RecBCD (Lane 3) and incubated for a further 5 min showing that sufficient cofactors were present.

    Article Snippet: ATP, SAM and EcoRI were purchased from New England Biolabs.

    Techniques: Plasmid Preparation, Incubation, Countercurrent Chromatography

    ATP hydrolysis activity of EcoKI on various oligonucleotides duplexes as measured using the NADH-coupled enzyme assay. The assay contained 10 nM EcoKI, 5 nM of the appropriate DNA duplex given in Table 1 , 0.1 mM SAM and 2 mM ATP.

    Journal: Nucleic Acids Research

    Article Title: An investigation of the structural requirements for ATP hydrolysis and DNA cleavage by the EcoKI Type I DNA restriction and modification enzyme

    doi: 10.1093/nar/gkr480

    Figure Lengend Snippet: ATP hydrolysis activity of EcoKI on various oligonucleotides duplexes as measured using the NADH-coupled enzyme assay. The assay contained 10 nM EcoKI, 5 nM of the appropriate DNA duplex given in Table 1 , 0.1 mM SAM and 2 mM ATP.

    Article Snippet: ATP, SAM and EcoRI were purchased from New England Biolabs.

    Techniques: Activity Assay, Nadh Coupled Enzyme Assay

    RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.

    Journal: mBio

    Article Title: RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling

    doi: 10.1128/mBio.00833-16

    Figure Lengend Snippet: RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.

    Article Snippet: The reaction volume was 500 µl and included 1 mg to 2 mg total lysate protein, RNA (33 nM to 1 µM), and 2 mM AMP-PNP (Roche) or ATP (New England Biolabs).

    Techniques: Western Blot, Incubation, RNA Binding Assay, Protease Inhibitor

    RNA mod and RIG-I trypsin sensitivity. (A to D) Digestion of 293T cell lysate for 2 h in the presence of polyU/UC RNA with the indicated modifications or canonical nucleotides (can), at increasing polyU/UC RNA concentrations (0, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 nM), in the presence of 2 mM ATP or AMP-PNP. Data represent results of Western blotting for 55-kDa and 80-kDa fragments of RIG-I with anti-helicase antibody. (E) Digestions of 293T cell lysate performed for 1.5 h in the presence of AMP-PNP and polyU/UC RNA with the indicated modifications, at increasing concentrations (0, 33, 100, 300, and 900 nM), or mass equivalent of polyI:C.

    Journal: mBio

    Article Title: RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling

    doi: 10.1128/mBio.00833-16

    Figure Lengend Snippet: RNA mod and RIG-I trypsin sensitivity. (A to D) Digestion of 293T cell lysate for 2 h in the presence of polyU/UC RNA with the indicated modifications or canonical nucleotides (can), at increasing polyU/UC RNA concentrations (0, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 nM), in the presence of 2 mM ATP or AMP-PNP. Data represent results of Western blotting for 55-kDa and 80-kDa fragments of RIG-I with anti-helicase antibody. (E) Digestions of 293T cell lysate performed for 1.5 h in the presence of AMP-PNP and polyU/UC RNA with the indicated modifications, at increasing concentrations (0, 33, 100, 300, and 900 nM), or mass equivalent of polyI:C.

    Article Snippet: The reaction volume was 500 µl and included 1 mg to 2 mg total lysate protein, RNA (33 nM to 1 µM), and 2 mM AMP-PNP (Roche) or ATP (New England Biolabs).

    Techniques: Western Blot