dnase  (New England Biolabs)


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  • 99
    Name:
    DNase I RNase free
    Description:
    DNase I RNase free 5 000 units
    Catalog Number:
    m0303l
    Price:
    282
    Size:
    5 000 units
    Category:
    Deoxyribonucleases DNase
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    New England Biolabs dnase
    DNase I RNase free
    DNase I RNase free 5 000 units
    https://www.bioz.com/result/dnase/product/New England Biolabs
    Average 99 stars, based on 60 article reviews
    Price from $9.99 to $1999.99
    dnase - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) Product Images from "Holliday junction affinity of the base excision repair factor Endo III contributes to cholera toxin phage integration"

    Article Title: Holliday junction affinity of the base excision repair factor Endo III contributes to cholera toxin phage integration

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2012.219

    Endo III blocks XerC-catalysis on pseudo-HJ and displaces it from them. ( A ) Scheme of the suicide pseudo-HJ indicating the mismatch engineered to slow down re-ligation after XerC-cleavage. KMnO 4 sensitive residues in the XerC- and XerD-binding att P(+) arms are indicated by a star. ( B ) Resolution of att P(+)/ dif suicide pseudo-HJs. Legend as in Figure 4D . ( C ) DNase I protection and ( D ) KMnO 4 sensitivity assays of the att P(+)/ dif 1 pseudo-HJ substrate. The analysed strand was labelled on its 5′ end. A scheme of the analysed strand is drawn on the left of the gels. KMnO 4 sensitive residues in the XerC- and XerD-binding sites are indicated by a star.
    Figure Legend Snippet: Endo III blocks XerC-catalysis on pseudo-HJ and displaces it from them. ( A ) Scheme of the suicide pseudo-HJ indicating the mismatch engineered to slow down re-ligation after XerC-cleavage. KMnO 4 sensitive residues in the XerC- and XerD-binding att P(+) arms are indicated by a star. ( B ) Resolution of att P(+)/ dif suicide pseudo-HJs. Legend as in Figure 4D . ( C ) DNase I protection and ( D ) KMnO 4 sensitivity assays of the att P(+)/ dif 1 pseudo-HJ substrate. The analysed strand was labelled on its 5′ end. A scheme of the analysed strand is drawn on the left of the gels. KMnO 4 sensitive residues in the XerC- and XerD-binding sites are indicated by a star.

    Techniques Used: Ligation, Binding Assay

    2) Product Images from "Comparison of Blood RNA Extraction Methods Used for Gene Expression Profiling in Amyotrophic Lateral Sclerosis"

    Article Title: Comparison of Blood RNA Extraction Methods Used for Gene Expression Profiling in Amyotrophic Lateral Sclerosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0087508

    Representative Bioanalyzer electropherograms after initial RNA extractions. High quality RNA (RIN > 7.0) can be extracted from all three methods. PAXGENE however requires a DNase step as traces from initial extractions show genomic DNA contamination at high molecular weights and as a “shoulder” to the 28S peak (indicated by asterisks). Good quality RNA can be detected after globin depletion with both TEM and PAXGENE. However, PAXGENE samples showed consistently lower concentration levels, as indicated by the smaller 18S and 28S peaks (FU, fluorescent units). Traces are representative and from single samples from each extraction method.
    Figure Legend Snippet: Representative Bioanalyzer electropherograms after initial RNA extractions. High quality RNA (RIN > 7.0) can be extracted from all three methods. PAXGENE however requires a DNase step as traces from initial extractions show genomic DNA contamination at high molecular weights and as a “shoulder” to the 28S peak (indicated by asterisks). Good quality RNA can be detected after globin depletion with both TEM and PAXGENE. However, PAXGENE samples showed consistently lower concentration levels, as indicated by the smaller 18S and 28S peaks (FU, fluorescent units). Traces are representative and from single samples from each extraction method.

    Techniques Used: Transmission Electron Microscopy, Concentration Assay

    3) Product Images from "3′ IsomiR Species and DNA Contamination Influence Reliable Quantification of MicroRNAs by Stem-Loop Quantitative PCR"

    Article Title: 3′ IsomiR Species and DNA Contamination Influence Reliable Quantification of MicroRNAs by Stem-Loop Quantitative PCR

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106315

    DNA can serve as a template during miRNA detection. ( A ) False positive signal of DNA derives mainly from the reverse transcription reaction. Mature miR-1226-3p was tested in the indicated samples, with or without reverse transcription (RT). On the y-axis, dC t value is represented, calculated as the Ct difference between the examined samples and the gDNA of control HeLa_mir-33b cell line (Ct = 35,9). One C t difference represents about 2× higher detected mature miRNA level. ( B ) DNA contamination remains in total RNA samples during isolation by the widely used Trizol reagent. Total RNA samples were isolated from transiently transfected HeLa cells; the transfected plasmid DNA amounts are indicated. Samples were DNase treated and non-treated, then reverse transcribed and subjected to real-time PCR. Expression values relative to U6 snRNA are shown on the y-axis. Experiments were carried out with three replicates at least from three independent experiments; one representative experiment is shown, error bars represent standard deviations.
    Figure Legend Snippet: DNA can serve as a template during miRNA detection. ( A ) False positive signal of DNA derives mainly from the reverse transcription reaction. Mature miR-1226-3p was tested in the indicated samples, with or without reverse transcription (RT). On the y-axis, dC t value is represented, calculated as the Ct difference between the examined samples and the gDNA of control HeLa_mir-33b cell line (Ct = 35,9). One C t difference represents about 2× higher detected mature miRNA level. ( B ) DNA contamination remains in total RNA samples during isolation by the widely used Trizol reagent. Total RNA samples were isolated from transiently transfected HeLa cells; the transfected plasmid DNA amounts are indicated. Samples were DNase treated and non-treated, then reverse transcribed and subjected to real-time PCR. Expression values relative to U6 snRNA are shown on the y-axis. Experiments were carried out with three replicates at least from three independent experiments; one representative experiment is shown, error bars represent standard deviations.

    Techniques Used: Isolation, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing

    Related Articles

    Centrifugation:

    Article Title: Human U6 promoter drives stronger shRNA activity than its schistosome orthologue in Schistosoma mansoni and human fibrosarcoma cells
    Article Snippet: .. Culture supernatants were collected 48 hours later, filtered through 0.45 μm pore size membranes, the filtrate incubated with DNAse I (New England Biolabs, Ipswich, MA) to remove contaminating plasmids, and virions in culture supernatants recovered by centrifugation (Sorvall SS34 rotor, 50,000 g, 90 minutes, 4°C). .. Pelleted virions were resuspended in schistosomule wash medium (above), virion titers estimated using quantitative PCR , and aliquots of virions stored at -80°C until needed. pLNHXΔD70 was employed to produce control virions without shRNA encoding cassettes.

    Amplification:

    Article Title: Evidence that Altered Cis Element Spacing Affects PpsR Mediated Redox Control of Photosynthesis Gene Expression in Rubrivivax gelatinosus
    Article Snippet: 188-bp pucB promoter region and 194-bp crtI promoter region containing two TGT-N12 -ACA PpsR-binding palindromes were amplified using a 6-FAM-labeled primer pucBfor ( 5’-AGAGCTCCCGAGGCCCGC-3’ ) and a 5-HEX-labeled primer pucBrev ( 5’-ACCAGTTTCCGTGCTCGACC-3’ ), a 6-FAM-labeled primer crtIfor ( 5’-ATCGGAGGGAACCAGGTGAT-3’ ) and a 5-HEX-labeled primer crtIrev ( 5’-ATCCTGCTTTCCGGCCGCG-3’ ), respectively. .. Initial binding reaction mixtures were incubated for 30 min at 22°C followed by a 15 min DNase I digestion that was initiated by adding 3 μl of DNase I (New England Biolabs) at an approximately 1:100 dilution (0.02 units/μl) in a footprint binding buffer, which gave partial probe digestion.

    Article Title: Chromatin remodeling mediated by the FOXA1/A2 transcription factors activates CFTR expression in intestinal epithelial cells
    Article Snippet: .. DNase I sensitivity was quantified relative to undigested DNA for each primer set, and shown relative to digestion of a non-DNase I hypersensitive amplicon, DHS1+5 kb, for each DNase I sample. .. The intron 3 (405 + 13.1 kb), DHS10(a,b), and DHS11 fragments were PCR amplified and cloned into the pGL3B luciferase vector (Promega) containing the 787 bp minimal CFTR promoter , using primers listed in , and sequence verified.

    Synthesized:

    Article Title: Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in Mycobacterium tuberculosis
    Article Snippet: BssHII, NotI, XbaI, Antarctic phosphatase, MRI, avian myeloblastosis virus reverse transcriptase, DNase I, and T4 DNA ligase were obtained from New England Biolabs. .. Custom oligodeoxyribonucleotides were synthesized by Eurofins MWG/Operon.

    Lambda DNA Preparation:

    Article Title: Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori
    Article Snippet: .. TieA showed a non-specific endonuclease activity similar to DNase I as it cleaved both circular pUC19 and linearized lambda DNA, whereas Rv3131 (lane 6) as well as heat-inactivated TieA (lane 4) did not show any detectable DNA cleavage activity (Figure ). .. Furthermore, our results demonstrated a Ca2+ –Mg2+ dependent DNA cleavage activity of TieA, since EDTA and SDS markedly inhibited the cleavage activity of TieA (Figure , lanes 8, 9, 17, 18).

    Construct:

    Article Title: Human U6 promoter drives stronger shRNA activity than its schistosome orthologue in Schistosoma mansoni and human fibrosarcoma cells
    Article Snippet: Plasmid constructs, prepared with a maxiprep kit (5 Prime, Gaithersburg, MD) were employed to produce the virions as described ( ; ). .. Culture supernatants were collected 48 hours later, filtered through 0.45 μm pore size membranes, the filtrate incubated with DNAse I (New England Biolabs, Ipswich, MA) to remove contaminating plasmids, and virions in culture supernatants recovered by centrifugation (Sorvall SS34 rotor, 50,000 g, 90 minutes, 4°C).

    Real-time Polymerase Chain Reaction:

    Article Title: Human U6 promoter drives stronger shRNA activity than its schistosome orthologue in Schistosoma mansoni and human fibrosarcoma cells
    Article Snippet: Culture supernatants were collected 48 hours later, filtered through 0.45 μm pore size membranes, the filtrate incubated with DNAse I (New England Biolabs, Ipswich, MA) to remove contaminating plasmids, and virions in culture supernatants recovered by centrifugation (Sorvall SS34 rotor, 50,000 g, 90 minutes, 4°C). .. Pelleted virions were resuspended in schistosomule wash medium (above), virion titers estimated using quantitative PCR , and aliquots of virions stored at -80°C until needed. pLNHXΔD70 was employed to produce control virions without shRNA encoding cassettes.

    Incubation:

    Article Title: A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA
    Article Snippet: Pellets were suspended in 50 µl NEB buffer 4 and 10 U Taq I (NEB) and were allowed to incubate at 65°C for 16 h. Taq I was inactivated by heating to 80°C for 20 min followed by an incubation with 2.5 U of shrimp alkaline phosphatase (NEB) for 30 min. .. Pellets were suspended in 5 µl DNase I buffer and 0.2 U DNase I (NEB) and 0.2 U snake venom phosphodiesterase (Worthington) was added to the reactions.

    Article Title: Evidence that Altered Cis Element Spacing Affects PpsR Mediated Redox Control of Photosynthesis Gene Expression in Rubrivivax gelatinosus
    Article Snippet: .. Initial binding reaction mixtures were incubated for 30 min at 22°C followed by a 15 min DNase I digestion that was initiated by adding 3 μl of DNase I (New England Biolabs) at an approximately 1:100 dilution (0.02 units/μl) in a footprint binding buffer, which gave partial probe digestion. ..

    Article Title: Human U6 promoter drives stronger shRNA activity than its schistosome orthologue in Schistosoma mansoni and human fibrosarcoma cells
    Article Snippet: .. Culture supernatants were collected 48 hours later, filtered through 0.45 μm pore size membranes, the filtrate incubated with DNAse I (New England Biolabs, Ipswich, MA) to remove contaminating plasmids, and virions in culture supernatants recovered by centrifugation (Sorvall SS34 rotor, 50,000 g, 90 minutes, 4°C). .. Pelleted virions were resuspended in schistosomule wash medium (above), virion titers estimated using quantitative PCR , and aliquots of virions stored at -80°C until needed. pLNHXΔD70 was employed to produce control virions without shRNA encoding cassettes.

    Activity Assay:

    Article Title: A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA
    Article Snippet: Paragraph title: β-gt specificity and activity assay ... Pellets were suspended in 5 µl DNase I buffer and 0.2 U DNase I (NEB) and 0.2 U snake venom phosphodiesterase (Worthington) was added to the reactions.

    Article Title: Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori
    Article Snippet: .. TieA showed a non-specific endonuclease activity similar to DNase I as it cleaved both circular pUC19 and linearized lambda DNA, whereas Rv3131 (lane 6) as well as heat-inactivated TieA (lane 4) did not show any detectable DNA cleavage activity (Figure ). .. Furthermore, our results demonstrated a Ca2+ –Mg2+ dependent DNA cleavage activity of TieA, since EDTA and SDS markedly inhibited the cleavage activity of TieA (Figure , lanes 8, 9, 17, 18).

    Expressing:

    Article Title: A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease
    Article Snippet: .. Materials Restriction enzymes, nicking enzyme N.BstNB I, DNA polymerases, T4 ligase, T4 DNA kinase, β-agarase, λ Exonuclease, DNase I, the maltose-binding protein (MBP) protein fusion expression and purification system including plasmid pMAL-c2x, the host E.coli strains TB1 and ER2566, Factor Xa protease, genenase, the cruciform structure-containing plasmid pUC(AT), 2 logs DNA ladder and synthetic oligonucleotides were obtained from New England Biolabs Inc. (NEB). .. Plasmid pNB1 was a gift from Rebecca Kucera (NEB).

    Magnetic Resonance Imaging:

    Article Title: Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in Mycobacterium tuberculosis
    Article Snippet: .. BssHII, NotI, XbaI, Antarctic phosphatase, MRI, avian myeloblastosis virus reverse transcriptase, DNase I, and T4 DNA ligase were obtained from New England Biolabs. .. Taq polymerase and deoxyribonucleotides were obtained from GenScript Corporation.

    other:

    Article Title: The Principal Role of Ku in Telomere Length Maintenance Is Promotion of Est1 Association with Telomeres
    Article Snippet: Furthermore, the interaction between Ku and Est1 and Est2 was not mediated by DNA as treatment of lysates with DNase I had no impact on the co-immunoprecipitation efficiency ( and Figure S3 A).

    Article Title: Regulation of the vapBC-1 Toxin-Antitoxin Locus in Nontypeable Haemophilus influenzae
    Article Snippet: Reactions were quenched with 75 µL of DNase I stop solution (90% ethanol/220 mM sodium acetate/70 ng/µL yeast tRNA), mixed, and placed immediately in a dry ice/ethanol bath for 30 min.

    Polymerase Chain Reaction:

    Article Title: Evidence that Altered Cis Element Spacing Affects PpsR Mediated Redox Control of Photosynthesis Gene Expression in Rubrivivax gelatinosus
    Article Snippet: Then, the PCR fragment was extracted and purified from 1.5% agarose gel in TAE buffer using QIAquick Gel Extraction Kit (QIAGEN). .. Initial binding reaction mixtures were incubated for 30 min at 22°C followed by a 15 min DNase I digestion that was initiated by adding 3 μl of DNase I (New England Biolabs) at an approximately 1:100 dilution (0.02 units/μl) in a footprint binding buffer, which gave partial probe digestion.

    Binding Assay:

    Article Title: Evidence that Altered Cis Element Spacing Affects PpsR Mediated Redox Control of Photosynthesis Gene Expression in Rubrivivax gelatinosus
    Article Snippet: .. Initial binding reaction mixtures were incubated for 30 min at 22°C followed by a 15 min DNase I digestion that was initiated by adding 3 μl of DNase I (New England Biolabs) at an approximately 1:100 dilution (0.02 units/μl) in a footprint binding buffer, which gave partial probe digestion. ..

    Fluorescence:

    Article Title: In-Cell RNA Hydrolysis Assay: A Method for the Determination of the RNase Activity of Potential RNases
    Article Snippet: .. The fluorescence intensity of RiboGreen was similar in the presence and absence of DNase I (Fig. f). .. DNA molecules packaged into chromatin in the nucleus would not be attacked by a protein with DNA- and RNA-hydrolyzing activities, such as 3D8 scFv antibody; therefore, the majority of RiboGreen fluorescence in this assay can be attributed to RNA, not to DNA.

    Article Title: Evidence that Altered Cis Element Spacing Affects PpsR Mediated Redox Control of Photosynthesis Gene Expression in Rubrivivax gelatinosus
    Article Snippet: Individual footprint reactions were initiated with a 22 μl binding reaction mixture that contained 12.5 mM HEPES (pH 7.8), 5 mM K-acetate (pH 8.0), 2.5 mM Mg-acetate, 1 mM CaCl2 , 12.5 μg/ml bovine serum albumin [ ], 0.3 mg/ml heparin, 200 nM of fluorescence-labeled DNA probe and various amounts of purified protein. .. Initial binding reaction mixtures were incubated for 30 min at 22°C followed by a 15 min DNase I digestion that was initiated by adding 3 μl of DNase I (New England Biolabs) at an approximately 1:100 dilution (0.02 units/μl) in a footprint binding buffer, which gave partial probe digestion.

    Article Title: In-Cell RNA Hydrolysis Assay: A Method for the Determination of the RNase Activity of Potential RNases
    Article Snippet: .. The fluorescence intensity of RiboGreen in the conditioned medium of cells treated with RNase A or 3D8 scFv antibody was similar in the presence and absence of DNase I. ..

    Labeling:

    Article Title: Evidence that Altered Cis Element Spacing Affects PpsR Mediated Redox Control of Photosynthesis Gene Expression in Rubrivivax gelatinosus
    Article Snippet: DNase I footprint assay Fluorescently labeled DNA probes containing either the pucB or crtI promoter regions were prepared by PCR amplification. .. Initial binding reaction mixtures were incubated for 30 min at 22°C followed by a 15 min DNase I digestion that was initiated by adding 3 μl of DNase I (New England Biolabs) at an approximately 1:100 dilution (0.02 units/μl) in a footprint binding buffer, which gave partial probe digestion.

    Purification:

    Article Title: A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA
    Article Snippet: Reactions were terminated by the addition of 10 µl Stop Buffer [20 µg Protenase K, 50 mM Tris–HCl (pH 8.0), 100 mM EDTA (pH 8.0), 2% (w/v) SDS] followed by incubation at 42°C for 60 min. Oligonucleotides were purified by phenol:CHCl3 :IAA extraction followed by an ethanol precipitation. .. Pellets were suspended in 5 µl DNase I buffer and 0.2 U DNase I (NEB) and 0.2 U snake venom phosphodiesterase (Worthington) was added to the reactions.

    Article Title: Evidence that Altered Cis Element Spacing Affects PpsR Mediated Redox Control of Photosynthesis Gene Expression in Rubrivivax gelatinosus
    Article Snippet: Individual footprint reactions were initiated with a 22 μl binding reaction mixture that contained 12.5 mM HEPES (pH 7.8), 5 mM K-acetate (pH 8.0), 2.5 mM Mg-acetate, 1 mM CaCl2 , 12.5 μg/ml bovine serum albumin [ ], 0.3 mg/ml heparin, 200 nM of fluorescence-labeled DNA probe and various amounts of purified protein. .. Initial binding reaction mixtures were incubated for 30 min at 22°C followed by a 15 min DNase I digestion that was initiated by adding 3 μl of DNase I (New England Biolabs) at an approximately 1:100 dilution (0.02 units/μl) in a footprint binding buffer, which gave partial probe digestion.

    Article Title: A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease
    Article Snippet: .. Materials Restriction enzymes, nicking enzyme N.BstNB I, DNA polymerases, T4 ligase, T4 DNA kinase, β-agarase, λ Exonuclease, DNase I, the maltose-binding protein (MBP) protein fusion expression and purification system including plasmid pMAL-c2x, the host E.coli strains TB1 and ER2566, Factor Xa protease, genenase, the cruciform structure-containing plasmid pUC(AT), 2 logs DNA ladder and synthetic oligonucleotides were obtained from New England Biolabs Inc. (NEB). .. Plasmid pNB1 was a gift from Rebecca Kucera (NEB).

    Plasmid Preparation:

    Article Title: A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease
    Article Snippet: .. Materials Restriction enzymes, nicking enzyme N.BstNB I, DNA polymerases, T4 ligase, T4 DNA kinase, β-agarase, λ Exonuclease, DNase I, the maltose-binding protein (MBP) protein fusion expression and purification system including plasmid pMAL-c2x, the host E.coli strains TB1 and ER2566, Factor Xa protease, genenase, the cruciform structure-containing plasmid pUC(AT), 2 logs DNA ladder and synthetic oligonucleotides were obtained from New England Biolabs Inc. (NEB). .. Plasmid pNB1 was a gift from Rebecca Kucera (NEB).

    Article Title: Human U6 promoter drives stronger shRNA activity than its schistosome orthologue in Schistosoma mansoni and human fibrosarcoma cells
    Article Snippet: In brief, GP2-293 cells were co-transfected with the MLV encoding plasmid ( ) along with pVSVG, which encodes vesicular stomatitis virus glycoprotein (the pseudotyping envelope) using liposomes (Lipofectamine 2000, Invitrogen). .. Culture supernatants were collected 48 hours later, filtered through 0.45 μm pore size membranes, the filtrate incubated with DNAse I (New England Biolabs, Ipswich, MA) to remove contaminating plasmids, and virions in culture supernatants recovered by centrifugation (Sorvall SS34 rotor, 50,000 g, 90 minutes, 4°C).

    shRNA:

    Article Title: Human U6 promoter drives stronger shRNA activity than its schistosome orthologue in Schistosoma mansoni and human fibrosarcoma cells
    Article Snippet: Culture supernatants were collected 48 hours later, filtered through 0.45 μm pore size membranes, the filtrate incubated with DNAse I (New England Biolabs, Ipswich, MA) to remove contaminating plasmids, and virions in culture supernatants recovered by centrifugation (Sorvall SS34 rotor, 50,000 g, 90 minutes, 4°C). .. Pelleted virions were resuspended in schistosomule wash medium (above), virion titers estimated using quantitative PCR , and aliquots of virions stored at -80°C until needed. pLNHXΔD70 was employed to produce control virions without shRNA encoding cassettes.

    Agarose Gel Electrophoresis:

    Article Title: Evidence that Altered Cis Element Spacing Affects PpsR Mediated Redox Control of Photosynthesis Gene Expression in Rubrivivax gelatinosus
    Article Snippet: Then, the PCR fragment was extracted and purified from 1.5% agarose gel in TAE buffer using QIAquick Gel Extraction Kit (QIAGEN). .. Initial binding reaction mixtures were incubated for 30 min at 22°C followed by a 15 min DNase I digestion that was initiated by adding 3 μl of DNase I (New England Biolabs) at an approximately 1:100 dilution (0.02 units/μl) in a footprint binding buffer, which gave partial probe digestion.

    Ethanol Precipitation:

    Article Title: A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA
    Article Snippet: DNA was cleaned by phenol:CHCl3 :IAA (isoamyl alcohol) extraction followed by an ethanol precipitation. .. Pellets were suspended in 5 µl DNase I buffer and 0.2 U DNase I (NEB) and 0.2 U snake venom phosphodiesterase (Worthington) was added to the reactions.

    Gel Extraction:

    Article Title: Evidence that Altered Cis Element Spacing Affects PpsR Mediated Redox Control of Photosynthesis Gene Expression in Rubrivivax gelatinosus
    Article Snippet: Then, the PCR fragment was extracted and purified from 1.5% agarose gel in TAE buffer using QIAquick Gel Extraction Kit (QIAGEN). .. Initial binding reaction mixtures were incubated for 30 min at 22°C followed by a 15 min DNase I digestion that was initiated by adding 3 μl of DNase I (New England Biolabs) at an approximately 1:100 dilution (0.02 units/μl) in a footprint binding buffer, which gave partial probe digestion.

    Variant Assay:

    Article Title: Virus-Inspired Membrane Encapsulation of DNA Nanostructures To Achieve In Vivo Stability
    Article Snippet: .. Ten units of DNase I were added to 3× replicates of each variant, and an equivalent volume of DNase I buffer was added to 3× replicates as negative controls. .. All samples were incubated for 24 h at 37 °C on a Tetrad 2 Peltier thermocycler.

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  • 99
    New England Biolabs ultrapure dnase i
    Mutating of the major C-terminal CKII sites does not affect interaction of CTCF with c- myc CTSs assayed by EMSAs (A), methylation interference (B), and <t>DNase</t> I footprinting (C). Lanes: 1, full-length (FL) wtCTCF; 2, CTCF/2-mut; 3, CTCF/4-mut; 4, 11ZF protein. See the text for more details. F, free probe; B, protein-bound probe.
    Ultrapure Dnase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ultrapure dnase i/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ultrapure dnase i - by Bioz Stars, 2020-04
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    86
    New England Biolabs dnase i treated dna
    A specifically positioned nucleosome covers the promoter of HPV-16. (A) The HPV-16 LCR cloned in pHPV-16-Luc was assembled into chromatin with Drosophila S190 extracts and treated with increasing amounts of <t>DNase</t> I, and the resulting fragments were purified and assayed by primer extension. Lanes 2 and 3, footprints originating from two nucleosomes that overlap with the promoter and the enhancer (large and small oval shapes on the right). Weak 10-bp-spaced bands (filled stars) indicate <t>DNase</t> I accessibility due to the rotational phasing of the nucleosomes, and a strong hypersensitive site (open star) suggests the center of the dyad symmetry of the nucleosome. As controls, lane 1 shows DNase I treatment of free HPV-16 LCR DNA and the left side of the panel indicates a sequencing ladder. Lane M, size marker with bands at 500, 400, 300, 200, and 100 bp. Symbols and nucleotides on the left identify the four cis -responsive elements of the E6 promoter, namely, binding sites for Sp1, the viral factor E2, and TBP. (B) Footprint obtained in a similar experiment and permitting similar interpretations. It highlights the 10-bp periodicity but does not permit clear mapping of the extent of nucleosomal protection.
    Dnase I Treated Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i treated dna/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dnase i treated dna - by Bioz Stars, 2020-04
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    95
    New England Biolabs bovine dnase i
    Wild-type or mutant HducCdtB proteins purified under denaturing conditions present a similar plasmid digestion activity. A. SDS-PAGE analysis of WT and D273R (DR) HducCdtB proteins purified from E . coli NiCo21 (DE3) under denaturing conditions. B. Kinetics and CdtB concentration effect on plasmid digestion assay with WT or D273R HducCdtB. Agarose gel electrophoresis and quantification of supercoiled plasmid (250 ng) incubated in presence of the indicated concentration of WT or D273R HducCdtB or bovine <t>DNase</t> I for the indicated times. C. Size exclusion chromatography (SEC) of WT HducCdtB. The curve represents the absorbance at 280 nm of the fractions eluted from the SEC column. The fractions used for further analysis are shown with black arrows. The selected fractions were analysed by Western Blot with anti-HIS antibody. The peak fractions from WT and D273R (DR) HducCdtB were analysed by Western Blot with anti-HIS antibody. D. Plasmid digestion assay with WT or D273R HducCdtB purified from SEC. Agarose gel electrophoresis and quantification of supercoiled plasmid (125 ng) incubated in presence of 1 μg of WT (D4) or D273R (D2) HducCdtB or in control conditions (no protein, with 2 ng of bovine DNase I or with 1 μg of CdtC) for 7 h in presence or in absence of Mg2+/Ca2+ buffer. B and D: arrows indicate plasmid conformation, either relaxed (R), linear (L) or supercoiled (S). For quantifications, the amount of each plasmid conformation is expressed as proportion of the total plasmid content. Results present the mean ± SD of at least three independent experiments, except for the D4 and D2 fractions without ions which were replicated twice; statistical differences were analysed between every conditions and only mutant vs WT comparisons are shown (ns: not significant).
    Bovine Dnase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 1 article reviews
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    86
    New England Biolabs bovine pancreas recombinant dnase i
    Transmission electron microscopy analysis of NETs. Human neutrophils were seeded in untreated 35-mm culture dishes and treated with 100 ng/ml LPS ( a–f ) or PMA ( g, h ) for 90 min at 37°C and then processed for transmission electron microscopy as described in Materials and Methods. a The arrows indicate NETforming cells with undetectable nuclear envelope or condensed chromatin. b Magnification of the area selected in a. The arrow shows partial preservation of the plasma membrane. c–f NETs usually contain vesicular membranes (arrowhead in e ) and granular structures (arrow in e ). d–f Magnifications of the areas selected in c–e , respectively. f Extracellular DNA fibers appear as a filamentous material (arrowheads) containing granular structures (arrows). g, h Analysis of cross-sections of NETs showing that the characteristic filamentous material adjacent to a PMAstimulated neutrophil ( g ) is completely dismantled by treatment with <t>DNase</t> I ( h ).
    Bovine Pancreas Recombinant Dnase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mutating of the major C-terminal CKII sites does not affect interaction of CTCF with c- myc CTSs assayed by EMSAs (A), methylation interference (B), and DNase I footprinting (C). Lanes: 1, full-length (FL) wtCTCF; 2, CTCF/2-mut; 3, CTCF/4-mut; 4, 11ZF protein. See the text for more details. F, free probe; B, protein-bound probe.

    Journal: Molecular and Cellular Biology

    Article Title: Functional Phosphorylation Sites in the C-Terminal Region of the Multivalent Multifunctional Transcriptional Factor CTCF

    doi: 10.1128/MCB.21.6.2221-2234.2001

    Figure Lengend Snippet: Mutating of the major C-terminal CKII sites does not affect interaction of CTCF with c- myc CTSs assayed by EMSAs (A), methylation interference (B), and DNase I footprinting (C). Lanes: 1, full-length (FL) wtCTCF; 2, CTCF/2-mut; 3, CTCF/4-mut; 4, 11ZF protein. See the text for more details. F, free probe; B, protein-bound probe.

    Article Snippet: For DNase I footprinting, 1 μl (1 Kunitz unit) of ultrapure DNase I (New England Biolabs) dissolved in phosphate-buffered saline supplemented with 1 mM CaCl2 and 1 mM MgCl2 was added to each of 10 32 P-DNA-CTCF binding mixtures, with 20-μl final volumes prepared as described for EMSA reactions, mixed at room temperature for 30 s, and immediately loaded on EMSA gels already running at 300 cV.

    Techniques: Methylation, Footprinting

    A specifically positioned nucleosome covers the promoter of HPV-16. (A) The HPV-16 LCR cloned in pHPV-16-Luc was assembled into chromatin with Drosophila S190 extracts and treated with increasing amounts of DNase I, and the resulting fragments were purified and assayed by primer extension. Lanes 2 and 3, footprints originating from two nucleosomes that overlap with the promoter and the enhancer (large and small oval shapes on the right). Weak 10-bp-spaced bands (filled stars) indicate DNase I accessibility due to the rotational phasing of the nucleosomes, and a strong hypersensitive site (open star) suggests the center of the dyad symmetry of the nucleosome. As controls, lane 1 shows DNase I treatment of free HPV-16 LCR DNA and the left side of the panel indicates a sequencing ladder. Lane M, size marker with bands at 500, 400, 300, 200, and 100 bp. Symbols and nucleotides on the left identify the four cis -responsive elements of the E6 promoter, namely, binding sites for Sp1, the viral factor E2, and TBP. (B) Footprint obtained in a similar experiment and permitting similar interpretations. It highlights the 10-bp periodicity but does not permit clear mapping of the extent of nucleosomal protection.

    Journal: Journal of Virology

    Article Title: The Chromatin Structure of the Long Control Region of Human Papillomavirus Type 16 Represses Viral Oncoprotein Expression

    doi:

    Figure Lengend Snippet: A specifically positioned nucleosome covers the promoter of HPV-16. (A) The HPV-16 LCR cloned in pHPV-16-Luc was assembled into chromatin with Drosophila S190 extracts and treated with increasing amounts of DNase I, and the resulting fragments were purified and assayed by primer extension. Lanes 2 and 3, footprints originating from two nucleosomes that overlap with the promoter and the enhancer (large and small oval shapes on the right). Weak 10-bp-spaced bands (filled stars) indicate DNase I accessibility due to the rotational phasing of the nucleosomes, and a strong hypersensitive site (open star) suggests the center of the dyad symmetry of the nucleosome. As controls, lane 1 shows DNase I treatment of free HPV-16 LCR DNA and the left side of the panel indicates a sequencing ladder. Lane M, size marker with bands at 500, 400, 300, 200, and 100 bp. Symbols and nucleotides on the left identify the four cis -responsive elements of the E6 promoter, namely, binding sites for Sp1, the viral factor E2, and TBP. (B) Footprint obtained in a similar experiment and permitting similar interpretations. It highlights the 10-bp periodicity but does not permit clear mapping of the extent of nucleosomal protection.

    Article Snippet: DNase I-treated DNA was purified by standard procedures and used as a template for the extension reaction with Vent (exo-) polymerase (New England Biolabs).

    Techniques: Clone Assay, Purification, Sequencing, Marker, Binding Assay

    A specifically positioned nucleosome covers the replication origin of HPV-18 and extends into E6 promoter sequences. The cloned HPV-18 LCR was assembled into chromatin with Drosophila S190 extracts and treated with increasing amounts of DNase I, and the resulting fragments were assayed by primer extension. Lanes 3 to 7 show, with increasing DNase I treatment, the footprint of a nucleosome overlapping the replication origin (distal E2 and E1 binding site), indicated by a large hatched oval on the right. Weak 10-bp-spaced bands (filled stars) indicate DNase I accessibility due to the rotational phasing of the nucleosomal organization, and a strong hypersensitivity site (open star) suggests the center of the dyad symmetry of the nucleosome. As controls, lanes 1 and 2 show DNase I treatment of free HPV-18 LCR DNA, and the left side shows a sequencing ladder of this sequence. Lane M, size marker with bands at 500, 400, 300, 200, and 100 bp. Symbols and nucleotides on the right identify the third E2 binding site from the E6 promoter, the E1 binding site, and one of the four cis -responsive elements of the E6 promoter, namely, the binding site for Sp1.

    Journal: Journal of Virology

    Article Title: The Chromatin Structure of the Long Control Region of Human Papillomavirus Type 16 Represses Viral Oncoprotein Expression

    doi:

    Figure Lengend Snippet: A specifically positioned nucleosome covers the replication origin of HPV-18 and extends into E6 promoter sequences. The cloned HPV-18 LCR was assembled into chromatin with Drosophila S190 extracts and treated with increasing amounts of DNase I, and the resulting fragments were assayed by primer extension. Lanes 3 to 7 show, with increasing DNase I treatment, the footprint of a nucleosome overlapping the replication origin (distal E2 and E1 binding site), indicated by a large hatched oval on the right. Weak 10-bp-spaced bands (filled stars) indicate DNase I accessibility due to the rotational phasing of the nucleosomal organization, and a strong hypersensitivity site (open star) suggests the center of the dyad symmetry of the nucleosome. As controls, lanes 1 and 2 show DNase I treatment of free HPV-18 LCR DNA, and the left side shows a sequencing ladder of this sequence. Lane M, size marker with bands at 500, 400, 300, 200, and 100 bp. Symbols and nucleotides on the right identify the third E2 binding site from the E6 promoter, the E1 binding site, and one of the four cis -responsive elements of the E6 promoter, namely, the binding site for Sp1.

    Article Snippet: DNase I-treated DNA was purified by standard procedures and used as a template for the extension reaction with Vent (exo-) polymerase (New England Biolabs).

    Techniques: Clone Assay, Binding Assay, Sequencing, Marker

    Wild-type or mutant HducCdtB proteins purified under denaturing conditions present a similar plasmid digestion activity. A. SDS-PAGE analysis of WT and D273R (DR) HducCdtB proteins purified from E . coli NiCo21 (DE3) under denaturing conditions. B. Kinetics and CdtB concentration effect on plasmid digestion assay with WT or D273R HducCdtB. Agarose gel electrophoresis and quantification of supercoiled plasmid (250 ng) incubated in presence of the indicated concentration of WT or D273R HducCdtB or bovine DNase I for the indicated times. C. Size exclusion chromatography (SEC) of WT HducCdtB. The curve represents the absorbance at 280 nm of the fractions eluted from the SEC column. The fractions used for further analysis are shown with black arrows. The selected fractions were analysed by Western Blot with anti-HIS antibody. The peak fractions from WT and D273R (DR) HducCdtB were analysed by Western Blot with anti-HIS antibody. D. Plasmid digestion assay with WT or D273R HducCdtB purified from SEC. Agarose gel electrophoresis and quantification of supercoiled plasmid (125 ng) incubated in presence of 1 μg of WT (D4) or D273R (D2) HducCdtB or in control conditions (no protein, with 2 ng of bovine DNase I or with 1 μg of CdtC) for 7 h in presence or in absence of Mg2+/Ca2+ buffer. B and D: arrows indicate plasmid conformation, either relaxed (R), linear (L) or supercoiled (S). For quantifications, the amount of each plasmid conformation is expressed as proportion of the total plasmid content. Results present the mean ± SD of at least three independent experiments, except for the D4 and D2 fractions without ions which were replicated twice; statistical differences were analysed between every conditions and only mutant vs WT comparisons are shown (ns: not significant).

    Journal: PLoS ONE

    Article Title: Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not

    doi: 10.1371/journal.pone.0214313

    Figure Lengend Snippet: Wild-type or mutant HducCdtB proteins purified under denaturing conditions present a similar plasmid digestion activity. A. SDS-PAGE analysis of WT and D273R (DR) HducCdtB proteins purified from E . coli NiCo21 (DE3) under denaturing conditions. B. Kinetics and CdtB concentration effect on plasmid digestion assay with WT or D273R HducCdtB. Agarose gel electrophoresis and quantification of supercoiled plasmid (250 ng) incubated in presence of the indicated concentration of WT or D273R HducCdtB or bovine DNase I for the indicated times. C. Size exclusion chromatography (SEC) of WT HducCdtB. The curve represents the absorbance at 280 nm of the fractions eluted from the SEC column. The fractions used for further analysis are shown with black arrows. The selected fractions were analysed by Western Blot with anti-HIS antibody. The peak fractions from WT and D273R (DR) HducCdtB were analysed by Western Blot with anti-HIS antibody. D. Plasmid digestion assay with WT or D273R HducCdtB purified from SEC. Agarose gel electrophoresis and quantification of supercoiled plasmid (125 ng) incubated in presence of 1 μg of WT (D4) or D273R (D2) HducCdtB or in control conditions (no protein, with 2 ng of bovine DNase I or with 1 μg of CdtC) for 7 h in presence or in absence of Mg2+/Ca2+ buffer. B and D: arrows indicate plasmid conformation, either relaxed (R), linear (L) or supercoiled (S). For quantifications, the amount of each plasmid conformation is expressed as proportion of the total plasmid content. Results present the mean ± SD of at least three independent experiments, except for the D4 and D2 fractions without ions which were replicated twice; statistical differences were analysed between every conditions and only mutant vs WT comparisons are shown (ns: not significant).

    Article Snippet: Bovine DNase I (New England Biolabs) was used as positive control.

    Techniques: Mutagenesis, Purification, Plasmid Preparation, Activity Assay, SDS Page, Concentration Assay, Agarose Gel Electrophoresis, Incubation, Size-exclusion Chromatography, Western Blot

    DNA damage induction in HeLa cells transfected with purified CdtB. A. Representative images of γH2AX immunofluorescence and DAPI staining from HeLa cells transfected with 50 nM of HducCdtB WT (fractions C2, C11, D4, E5) or D273R (fraction D2) after SEC for 24 h. Scale bar: 20 μm. B. Representative images of γH2AX immunofluorescence and DAPI staining from HeLa cells transfected with 50 nM of WT or D273R HducCdtB before SEC, with 200 nM of DNase I or with 120 nM of HducCdtC for 24 h. Scale bar: 20 μm. C. Representative images of γH2AX immunofluorescence and DAPI staining from HeLa cells transfected with 120 nM of EcolCdtB WT or H153A for 14 h. Scale bar: 20 μm. D. Representative images of γH2AX and 53BP1 immunofluorescence and DAPI staining from HeLa cells transfected with 5 nM of WT or mutant (Hduc D273R or Ecol H153A) CdtB for 14 h. Scale bar: 20 μm. E. Dose-response analysis of γH2AX induction after CdtB transfection. Quantification of γH2AX signal in HeLa cells left untransfected (NT) or transfected with the indicated concentration of WT or mutant (Hduc D273R or Ecol H153A) CdtB or negative controls (HducCdtC or DNase I) for 14 h, represented as the mean fluorescence intensity per cell (normalised to 1 for the untreated condition) or as the proportion of γH2AX positive cells. Results present the mean ± SD of at least three independent experiments. Statistical differences were analysed between transfected and non-transfected conditions for each CdtB concentration (* P

    Journal: PLoS ONE

    Article Title: Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not

    doi: 10.1371/journal.pone.0214313

    Figure Lengend Snippet: DNA damage induction in HeLa cells transfected with purified CdtB. A. Representative images of γH2AX immunofluorescence and DAPI staining from HeLa cells transfected with 50 nM of HducCdtB WT (fractions C2, C11, D4, E5) or D273R (fraction D2) after SEC for 24 h. Scale bar: 20 μm. B. Representative images of γH2AX immunofluorescence and DAPI staining from HeLa cells transfected with 50 nM of WT or D273R HducCdtB before SEC, with 200 nM of DNase I or with 120 nM of HducCdtC for 24 h. Scale bar: 20 μm. C. Representative images of γH2AX immunofluorescence and DAPI staining from HeLa cells transfected with 120 nM of EcolCdtB WT or H153A for 14 h. Scale bar: 20 μm. D. Representative images of γH2AX and 53BP1 immunofluorescence and DAPI staining from HeLa cells transfected with 5 nM of WT or mutant (Hduc D273R or Ecol H153A) CdtB for 14 h. Scale bar: 20 μm. E. Dose-response analysis of γH2AX induction after CdtB transfection. Quantification of γH2AX signal in HeLa cells left untransfected (NT) or transfected with the indicated concentration of WT or mutant (Hduc D273R or Ecol H153A) CdtB or negative controls (HducCdtC or DNase I) for 14 h, represented as the mean fluorescence intensity per cell (normalised to 1 for the untreated condition) or as the proportion of γH2AX positive cells. Results present the mean ± SD of at least three independent experiments. Statistical differences were analysed between transfected and non-transfected conditions for each CdtB concentration (* P

    Article Snippet: Bovine DNase I (New England Biolabs) was used as positive control.

    Techniques: Transfection, Purification, Immunofluorescence, Staining, Size-exclusion Chromatography, Mutagenesis, Concentration Assay, Fluorescence

    Transmission electron microscopy analysis of NETs. Human neutrophils were seeded in untreated 35-mm culture dishes and treated with 100 ng/ml LPS ( a–f ) or PMA ( g, h ) for 90 min at 37°C and then processed for transmission electron microscopy as described in Materials and Methods. a The arrows indicate NETforming cells with undetectable nuclear envelope or condensed chromatin. b Magnification of the area selected in a. The arrow shows partial preservation of the plasma membrane. c–f NETs usually contain vesicular membranes (arrowhead in e ) and granular structures (arrow in e ). d–f Magnifications of the areas selected in c–e , respectively. f Extracellular DNA fibers appear as a filamentous material (arrowheads) containing granular structures (arrows). g, h Analysis of cross-sections of NETs showing that the characteristic filamentous material adjacent to a PMAstimulated neutrophil ( g ) is completely dismantled by treatment with DNase I ( h ).

    Journal: Journal of Innate Immunity

    Article Title: DNase I Inhibits a Late Phase of Reactive Oxygen Species Production in Neutrophils

    doi: 10.1159/000235860

    Figure Lengend Snippet: Transmission electron microscopy analysis of NETs. Human neutrophils were seeded in untreated 35-mm culture dishes and treated with 100 ng/ml LPS ( a–f ) or PMA ( g, h ) for 90 min at 37°C and then processed for transmission electron microscopy as described in Materials and Methods. a The arrows indicate NETforming cells with undetectable nuclear envelope or condensed chromatin. b Magnification of the area selected in a. The arrow shows partial preservation of the plasma membrane. c–f NETs usually contain vesicular membranes (arrowhead in e ) and granular structures (arrow in e ). d–f Magnifications of the areas selected in c–e , respectively. f Extracellular DNA fibers appear as a filamentous material (arrowheads) containing granular structures (arrows). g, h Analysis of cross-sections of NETs showing that the characteristic filamentous material adjacent to a PMAstimulated neutrophil ( g ) is completely dismantled by treatment with DNase I ( h ).

    Article Snippet: In these studies, we utilized 2 different sources of bovine pancreas recombinant DNase I [New England Biolab (DNase IA) and Worthington (DNase IB)].

    Techniques: Transmission Assay, Electron Microscopy, Preserving

    ROS production is attenuated by DNase I. Human neutrophils were stimulated with opsonized E. coli ( a ) or HKLM ( b )in the presence or absence of 100 U/ml DNase I. ROS production was measured as luminol-dependent chemiluminescence as described in Materials and Methods. Kinetic results are the mean ± SEM of 1 representative experiment performed in triplicate (left panels). Unstimulated samples showed no significant luminescence (data not shown). The right panels show the maximum chemiluminescence at the indicated time. Results are the mean ± SEM of 3 independent experiments with samples from different donors. Maximum chemiluminescence in samples treated with DNase I at 75 min but not at 30 min was significantly different from control (* p

    Journal: Journal of Innate Immunity

    Article Title: DNase I Inhibits a Late Phase of Reactive Oxygen Species Production in Neutrophils

    doi: 10.1159/000235860

    Figure Lengend Snippet: ROS production is attenuated by DNase I. Human neutrophils were stimulated with opsonized E. coli ( a ) or HKLM ( b )in the presence or absence of 100 U/ml DNase I. ROS production was measured as luminol-dependent chemiluminescence as described in Materials and Methods. Kinetic results are the mean ± SEM of 1 representative experiment performed in triplicate (left panels). Unstimulated samples showed no significant luminescence (data not shown). The right panels show the maximum chemiluminescence at the indicated time. Results are the mean ± SEM of 3 independent experiments with samples from different donors. Maximum chemiluminescence in samples treated with DNase I at 75 min but not at 30 min was significantly different from control (* p

    Article Snippet: In these studies, we utilized 2 different sources of bovine pancreas recombinant DNase I [New England Biolab (DNase IA) and Worthington (DNase IB)].

    Techniques:

    Localization of NADPH oxidase subunits on NETs. Neutrophils were stimulated with PMA (100 ng/ml), LPS (100 ng/ml) or HKLM for 90 min, and the NADPH oxidase subunits were detected on NETs by immunofluorescence confocal microscopy analysis as described in Materials and Methods.  a  Immunofluorescence analysis showing that p22 phox  is present on extracellular DNA fibers in fields where there is significant NET formation in neutrophils stimulated with phorbol ester (upper panels). The lower panels show a higher magnification of a field where p22 phox  can be detected in punctate structures on NETs. Arrows indicate p22 phox -specific staining on NETs; arrowheads indicate p22 phox -specific staining on intact neutrophils. Scale bars = 10 μm.  b  The upper and middle panels show localization of p67 phox  and p22 phox , respectively, on NETs detected using DAPI. NADPH oxidase subunits were detected in close proximity to the ectosome marker CD11b. Some of these structures are indicated with arrows. The lower panels show the magnification of the area highlighted on the middle panels. Scale bars = 10 μm.  c  Control antibodies do not recognize structures on NETs when used under the same experimental conditions. Scale bar = 10 μm.  d  Immunofluorescence analysis shows that the cytosolic subunit p47  phox  and the cytochrome b 558  component p22 phox  colocalize on punctate structures distributed on NETs. Some of these structures are indicated with arrows. Neither NETs nor NADPH oxidase subunits were detected extracellularly in the absence of stimuli. Scale bars = 10 μm.  e  Immunofluorescence analysis of the distribution of NADPH oxidase subunits on NETs in neutrophils stimulated with 100 ng/ml LPS for 90 min in the absence (upper panels) or presence (lower panels) of 100 U/ml DNase I. Scale bars = 10 μm.

    Journal: Journal of Innate Immunity

    Article Title: DNase I Inhibits a Late Phase of Reactive Oxygen Species Production in Neutrophils

    doi: 10.1159/000235860

    Figure Lengend Snippet: Localization of NADPH oxidase subunits on NETs. Neutrophils were stimulated with PMA (100 ng/ml), LPS (100 ng/ml) or HKLM for 90 min, and the NADPH oxidase subunits were detected on NETs by immunofluorescence confocal microscopy analysis as described in Materials and Methods. a Immunofluorescence analysis showing that p22 phox is present on extracellular DNA fibers in fields where there is significant NET formation in neutrophils stimulated with phorbol ester (upper panels). The lower panels show a higher magnification of a field where p22 phox can be detected in punctate structures on NETs. Arrows indicate p22 phox -specific staining on NETs; arrowheads indicate p22 phox -specific staining on intact neutrophils. Scale bars = 10 μm. b The upper and middle panels show localization of p67 phox and p22 phox , respectively, on NETs detected using DAPI. NADPH oxidase subunits were detected in close proximity to the ectosome marker CD11b. Some of these structures are indicated with arrows. The lower panels show the magnification of the area highlighted on the middle panels. Scale bars = 10 μm. c Control antibodies do not recognize structures on NETs when used under the same experimental conditions. Scale bar = 10 μm. d Immunofluorescence analysis shows that the cytosolic subunit p47 phox and the cytochrome b 558 component p22 phox colocalize on punctate structures distributed on NETs. Some of these structures are indicated with arrows. Neither NETs nor NADPH oxidase subunits were detected extracellularly in the absence of stimuli. Scale bars = 10 μm. e Immunofluorescence analysis of the distribution of NADPH oxidase subunits on NETs in neutrophils stimulated with 100 ng/ml LPS for 90 min in the absence (upper panels) or presence (lower panels) of 100 U/ml DNase I. Scale bars = 10 μm.

    Article Snippet: In these studies, we utilized 2 different sources of bovine pancreas recombinant DNase I [New England Biolab (DNase IA) and Worthington (DNase IB)].

    Techniques: Immunofluorescence, Confocal Microscopy, Staining, Marker

    Lack of inhibition of NADPH oxidase and MPO by DNase I treatment. a The NADPH oxidase total recombinant system was used to evaluate a possible effect of DNase I on the oxidase activity. To this end, recombinant proteins were incubated for 5 min to allow the assembly of the oxidase and were subsequently incubated in the presence of 100 U/ml DNase IA (New England Biolab) and DNase IB (Worthington) in their corresponding reaction buffers, in the absence of DNase I and DNase buffer (control) or in the presence of superoxide dismutase (SOD) for 10 min at 37°C. The production of superoxide anion was continuously monitored by cytochrome c reduction at 550 nm. The results (mean ± SEM) are representative of 2 independent experiments performed in triplicates. b Human MPO (60 ng/ml) was incubated in the presence or absence of DNase I (100 U/ml) for 1 h at 37°C. MPO activity was measured as described in Materials and Methods. The results are the mean ± SEM of 3 independent experiments performed in triplicates. No significant differences were found between DNase-I-treated and –untreated samples.

    Journal: Journal of Innate Immunity

    Article Title: DNase I Inhibits a Late Phase of Reactive Oxygen Species Production in Neutrophils

    doi: 10.1159/000235860

    Figure Lengend Snippet: Lack of inhibition of NADPH oxidase and MPO by DNase I treatment. a The NADPH oxidase total recombinant system was used to evaluate a possible effect of DNase I on the oxidase activity. To this end, recombinant proteins were incubated for 5 min to allow the assembly of the oxidase and were subsequently incubated in the presence of 100 U/ml DNase IA (New England Biolab) and DNase IB (Worthington) in their corresponding reaction buffers, in the absence of DNase I and DNase buffer (control) or in the presence of superoxide dismutase (SOD) for 10 min at 37°C. The production of superoxide anion was continuously monitored by cytochrome c reduction at 550 nm. The results (mean ± SEM) are representative of 2 independent experiments performed in triplicates. b Human MPO (60 ng/ml) was incubated in the presence or absence of DNase I (100 U/ml) for 1 h at 37°C. MPO activity was measured as described in Materials and Methods. The results are the mean ± SEM of 3 independent experiments performed in triplicates. No significant differences were found between DNase-I-treated and –untreated samples.

    Article Snippet: In these studies, we utilized 2 different sources of bovine pancreas recombinant DNase I [New England Biolab (DNase IA) and Worthington (DNase IB)].

    Techniques: Inhibition, Recombinant, Activity Assay, Incubation, IA