mnase  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    Name:
    Ambion RNase III
    Description:
    Ambion Escherichia coli ribonuclease III (RNase III; EC 3.1.24) is a double-stranded RNA (dsRNA) specific endoribonuclease. In E. coli, RNase III cleaves ribosomal RNA (rRNA) precursors during maturation of rRNA. The enzyme cleaves dsRNA into 12–15 bp dsRNA fragments with 2 to 3 nucleotide 3' overhangs, and 5' phosphate and 3' hydroxyl termini. The termini and overhangs of RNase III cleavage products are thus the same as those produced by Dicer in the eukaryotic RNAi pathway. Transfection of RNase III cleavage products can be used to induce RNAi in mammalian cells (patent pending).Unit Definition:One unit is defined as the amount of enzyme catalyzing the cleavage of 1 µg of 500 bp dsRNA substrate to approximately 12–30 bp fragments in 60 min at 37°C.
    Catalog Number:
    AM2290
    Price:
    None
    Applications:
    DNA & RNA Purification & Analysis|In Vitro Transcribed siRNA & Dicer siRNA|Nuclease Protection Assays|RNAi|RNAi, Epigenetics & Non-Coding RNA Research|Nucleic Acid Gel Electrophoresis & Blotting
    Size:
    250 units
    Category:
    Proteins, Enzymes, & Peptides, PCR & Cloning Enzymes, DNA⁄RNA Modifying Enzymes
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher mnase
    <t>DKC1-associated</t> RNAs in recombinant DKC1 complexes are resistant to extensive <t>MNase</t> digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010
    Ambion Escherichia coli ribonuclease III (RNase III; EC 3.1.24) is a double-stranded RNA (dsRNA) specific endoribonuclease. In E. coli, RNase III cleaves ribosomal RNA (rRNA) precursors during maturation of rRNA. The enzyme cleaves dsRNA into 12–15 bp dsRNA fragments with 2 to 3 nucleotide 3' overhangs, and 5' phosphate and 3' hydroxyl termini. The termini and overhangs of RNase III cleavage products are thus the same as those produced by Dicer in the eukaryotic RNAi pathway. Transfection of RNase III cleavage products can be used to induce RNAi in mammalian cells (patent pending).Unit Definition:One unit is defined as the amount of enzyme catalyzing the cleavage of 1 µg of 500 bp dsRNA substrate to approximately 12–30 bp fragments in 60 min at 37°C.
    https://www.bioz.com/result/mnase/product/Thermo Fisher
    Average 95 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    mnase - by Bioz Stars, 2019-12
    95/100 stars

    Images

    1) Product Images from "The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells"

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

    Journal: eLife

    doi: 10.7554/eLife.03573

    DKC1-associated RNAs in recombinant DKC1 complexes are resistant to extensive MNase digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010
    Figure Legend Snippet: DKC1-associated RNAs in recombinant DKC1 complexes are resistant to extensive MNase digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010

    Techniques Used: Recombinant, Purification, Labeling, Stable Transfection

    Micrococcal nuclease (MNase)-treated recombinant DKC1 complexes remain structurally intact. Recombinant wild-type (WT) and various mutant DKC1 complexes are mock treated (−) or digested extensively with MNase (+), and washed extensively to remove any dissociated RNAs prior to FLAG peptide elution. Eluted protein complexes are analyzed by Coomassie Blue staining. DOI: http://dx.doi.org/10.7554/eLife.03573.009
    Figure Legend Snippet: Micrococcal nuclease (MNase)-treated recombinant DKC1 complexes remain structurally intact. Recombinant wild-type (WT) and various mutant DKC1 complexes are mock treated (−) or digested extensively with MNase (+), and washed extensively to remove any dissociated RNAs prior to FLAG peptide elution. Eluted protein complexes are analyzed by Coomassie Blue staining. DOI: http://dx.doi.org/10.7554/eLife.03573.009

    Techniques Used: Recombinant, Mutagenesis, Staining

    MNase digestion moderately increases DKC1 coactivator activity. Mock (−) or MNase-treated (+) WT and Nop10 R34W DKC1 complexes are assayed in in vitro transcription reactions (over a fourfold concentration range) supplemented with OCT4, SOX2, recombinant XPC complex and SCC-B. DOI: http://dx.doi.org/10.7554/eLife.03573.011
    Figure Legend Snippet: MNase digestion moderately increases DKC1 coactivator activity. Mock (−) or MNase-treated (+) WT and Nop10 R34W DKC1 complexes are assayed in in vitro transcription reactions (over a fourfold concentration range) supplemented with OCT4, SOX2, recombinant XPC complex and SCC-B. DOI: http://dx.doi.org/10.7554/eLife.03573.011

    Techniques Used: Activity Assay, In Vitro, Concentration Assay, Recombinant

    2) Product Images from "Chromatin Structure and Transcription of the R1- and R2-Inserted rRNA Genes of Drosophila melanogaster"

    Article Title: Chromatin Structure and Transcription of the R1- and R2-Inserted rRNA Genes of Drosophila melanogaster

    Journal:

    doi: 10.1128/MCB.01409-06

    Digestion of different regions of the rDNA units with MNase. (A) Isolated 1- to 16-h embryonic nuclei were digested with MNase (0.5 U/μl) at 24°C for 0, 1, or 4 min (lanes 2 to 4 of each panel). Purified (protein-free) genomic DNA was
    Figure Legend Snippet: Digestion of different regions of the rDNA units with MNase. (A) Isolated 1- to 16-h embryonic nuclei were digested with MNase (0.5 U/μl) at 24°C for 0, 1, or 4 min (lanes 2 to 4 of each panel). Purified (protein-free) genomic DNA was

    Techniques Used: Isolation, Purification

    3) Product Images from "Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans"

    Article Title: Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000639

    Neutrophils release calprotectin by forming NETs. (A–F) Confocal images of human neutrophils without stimulation (A), after 0.5 h (B), 1 h (C), 2 h (D), 3 h (E) and 4 h (F) after activation. Samples were stained with antibodies specific for the calprotectin heteroduplex (red) and for MPO (green). DNA was stained with DRAQ5 (blue). Calprotectin localizes to the cytoplasm and partially to the nucleus (A, arrow). After stimulation for 0.5 h (B) the neutrophils flattened and formed numerous vacuoles. This reveals a granular staining for MPO and a more dispersed cytoplasmic staining for calprotectin. After stimulation for 1 h (C) the neutrophils round up slightly. The MPO and calprotectin stain partially overlap in the cytoplasm. After stimulation for 2 h (D), calprotectin, MPO and nuclear DNA start to colocalize in the decondensed nucleus (purple). After 3 h (E) and more so after 4 h (F) of stimulation, the cell membrane ruptures and calprotectin is released in NETs colocalizing with MPO and DNA. Scale bar = 10 µm; one experiment out of two is shown. (G–I) Subunits of calprotectin S100A8 and S100A9 are released after cell death during NET formation and not by degranulation. NET formation was induced with PMA and degranulation using formyl-met-leu-phe (f-MLP). (G) Neutrophil death was monitored by quantification of LDH activity in supernatants calculated as means±s.d. (n = 3). (H) Release of S100A8, S100A9, lactotransferrin (LTF) and myeloperoxidase (MPO) were analyzed by immunoblotting. one experiment out of two is shown. (I) Quantification of immunoblots using 2D densitometry analyzing S100A9 protein preparations from supernatants (lane 1), MNase-digested NETs (lane 2) and cell remnants indigestible for MNase (lane 3). Values were calculated as means±s.d. (n = 3) from one experiment out of two.
    Figure Legend Snippet: Neutrophils release calprotectin by forming NETs. (A–F) Confocal images of human neutrophils without stimulation (A), after 0.5 h (B), 1 h (C), 2 h (D), 3 h (E) and 4 h (F) after activation. Samples were stained with antibodies specific for the calprotectin heteroduplex (red) and for MPO (green). DNA was stained with DRAQ5 (blue). Calprotectin localizes to the cytoplasm and partially to the nucleus (A, arrow). After stimulation for 0.5 h (B) the neutrophils flattened and formed numerous vacuoles. This reveals a granular staining for MPO and a more dispersed cytoplasmic staining for calprotectin. After stimulation for 1 h (C) the neutrophils round up slightly. The MPO and calprotectin stain partially overlap in the cytoplasm. After stimulation for 2 h (D), calprotectin, MPO and nuclear DNA start to colocalize in the decondensed nucleus (purple). After 3 h (E) and more so after 4 h (F) of stimulation, the cell membrane ruptures and calprotectin is released in NETs colocalizing with MPO and DNA. Scale bar = 10 µm; one experiment out of two is shown. (G–I) Subunits of calprotectin S100A8 and S100A9 are released after cell death during NET formation and not by degranulation. NET formation was induced with PMA and degranulation using formyl-met-leu-phe (f-MLP). (G) Neutrophil death was monitored by quantification of LDH activity in supernatants calculated as means±s.d. (n = 3). (H) Release of S100A8, S100A9, lactotransferrin (LTF) and myeloperoxidase (MPO) were analyzed by immunoblotting. one experiment out of two is shown. (I) Quantification of immunoblots using 2D densitometry analyzing S100A9 protein preparations from supernatants (lane 1), MNase-digested NETs (lane 2) and cell remnants indigestible for MNase (lane 3). Values were calculated as means±s.d. (n = 3) from one experiment out of two.

    Techniques Used: Activation Assay, Staining, Activity Assay, Western Blot

    4) Product Images from "Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns"

    Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.450

    Release of nucleosomes and DAMPs from amino-acid-depleted HeLa cells. ( A ) An inverted microscopic image of HeLa cells in the condition of amino-acid depletion. Arrows designate dying HeLa cells. ( B ) Genomic sequences of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), Fas, cytochrome oxidase subunit 1 ( Co1 ) and ATP synthase subunit 6 ( ATP6 ) were PCR amplified from extracellularly released DNA, genomic or mitochondrial DNAs. ( C ) Inverted and fluorescent microscopic images were taken from amino-acid-deprived HeLa cells in the presence of SYTOX, a membrane-impermeable DNA dye. HeLa cells deprived of amino acids for 15 h were fluorescence stained with histone H1 or IL6 antibodies ( D ), histone H2A, H2B, H3 or H4 antibodies ( E ) or HMGB1, Hsp90 or ERp57 antibodies ( F ) in combination with histone H1 antibody and 4',6-diamidino-2-phenylindole (DAPI). ( G ) Amino-acid-deprived HeLa cells were stained with SYTOX to determine viability, fixed and stained with DAPI and histone H1 antibodies. ( H ) Amino-acid-deprived HeLa cells were untreated or treated with MNase (500 mU/ml) for 10 min. Released DNA was quantitated at the indicated times. Data from triplicate samples are presented as mean±S.D. ( I ) Conditioned media from amino-acid-deprived HeLa cells treated or untreated with MNase were western blotted with histone H1, 2B, H3, H4, IL6, ERp57, HMGB1 or Hsp90 antibodies. ( J ) Images captured every hour from live imaging of amino-acid-deprived HeLa cells with SYTOX (green) and DRAQ5, membrane-permeable DNA dye (red). ( K ) SYTOX fluorescent intensities were measured from circularized areas of live imaging of amino-acid-deprived cells in 5-min intervals. ( L ) TEM images of control cells ( L a) and amino-acid-deprived HeLa cells ( L b– L d). ( M ) Amino-acid-deprived HeLa cells were fluorescence stained with lamin and nuclear pore antibodies, or lamin antibody and wheat germ agglutinin (WGE)
    Figure Legend Snippet: Release of nucleosomes and DAMPs from amino-acid-depleted HeLa cells. ( A ) An inverted microscopic image of HeLa cells in the condition of amino-acid depletion. Arrows designate dying HeLa cells. ( B ) Genomic sequences of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), Fas, cytochrome oxidase subunit 1 ( Co1 ) and ATP synthase subunit 6 ( ATP6 ) were PCR amplified from extracellularly released DNA, genomic or mitochondrial DNAs. ( C ) Inverted and fluorescent microscopic images were taken from amino-acid-deprived HeLa cells in the presence of SYTOX, a membrane-impermeable DNA dye. HeLa cells deprived of amino acids for 15 h were fluorescence stained with histone H1 or IL6 antibodies ( D ), histone H2A, H2B, H3 or H4 antibodies ( E ) or HMGB1, Hsp90 or ERp57 antibodies ( F ) in combination with histone H1 antibody and 4',6-diamidino-2-phenylindole (DAPI). ( G ) Amino-acid-deprived HeLa cells were stained with SYTOX to determine viability, fixed and stained with DAPI and histone H1 antibodies. ( H ) Amino-acid-deprived HeLa cells were untreated or treated with MNase (500 mU/ml) for 10 min. Released DNA was quantitated at the indicated times. Data from triplicate samples are presented as mean±S.D. ( I ) Conditioned media from amino-acid-deprived HeLa cells treated or untreated with MNase were western blotted with histone H1, 2B, H3, H4, IL6, ERp57, HMGB1 or Hsp90 antibodies. ( J ) Images captured every hour from live imaging of amino-acid-deprived HeLa cells with SYTOX (green) and DRAQ5, membrane-permeable DNA dye (red). ( K ) SYTOX fluorescent intensities were measured from circularized areas of live imaging of amino-acid-deprived cells in 5-min intervals. ( L ) TEM images of control cells ( L a) and amino-acid-deprived HeLa cells ( L b– L d). ( M ) Amino-acid-deprived HeLa cells were fluorescence stained with lamin and nuclear pore antibodies, or lamin antibody and wheat germ agglutinin (WGE)

    Techniques Used: Genomic Sequencing, Polymerase Chain Reaction, Amplification, Fluorescence, Staining, Western Blot, Imaging, Transmission Electron Microscopy

    Related Articles

    Centrifugation:

    Article Title: Noncoding transcription influences the replication initiation program through chromatin regulation
    Article Snippet: Chromatin was then collected by centrifugation and resuspended in NP-buffer (0.5 mM spermidine, 1 mM β-ME, 0.075% NP-40, 50 mM NaCl, 10 mM Tris at pH 7.4, 5 mM MgCl2 , 1 mM CaCl2 ). .. MNase (Thermo Fisher Scientific) treatment was performed at a previously optimized concentration to have comparable intensity of both mono- and di-nucleosomes within and between the samples.

    Amplification:

    Article Title: Permissiveness of human hepatoma cell lines for HCV infection
    Article Snippet: Extracellular RNA from cell supernatants or intracellular HCV RNA from lysed cell pellets was treated with 4.5 units of S7 Micrococcal nuclease (Fermentas, Glen Burnie, MD) for 30 min at room temperature, and nuclease-treated RNA was isolated by the guanidine thiocyanate (GTC) method using 1.6 × GTC containing 2 μg of murine total liver RNA and following standard protocols [ ]. .. HCV, human GAPDH, murine GAPDH, human CD81, human SR-BI, human CLDN1, human OCLN and human ISG56 RNA levels were determined relative to standard curves comprised of serial dilutions of plasmids containing the JFH-1 HCV cDNA or the human GAPDH, murine GAPDH, human CD81, human SR-BI, human CLDN1, human OCLN and human ISG56 coding sequences, respectively.

    Picogreen Assay:

    Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns
    Article Snippet: Released DNA from dying cells were digested with 500 mU/ml MNase (Thermo Scientific) for 5 min. Nuclease activity was stopped with 5 mM EDTA and the culture supernatants were collected and stored at −20 °C until quantification. .. Released DNA from dying cells were digested with 500 mU/ml MNase (Thermo Scientific) for 5 min. Nuclease activity was stopped with 5 mM EDTA and the culture supernatants were collected and stored at −20 °C until quantification.

    Nuclease Assay:

    Article Title: Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity
    Article Snippet: Paragraph title: Micrococcal nuclease assay ... Samples were incubated with 1U of MNase (Thermo Scientific) for 0, 5, 25 minutes at 37 °C, and the reaction was stopped with 2 mM EDTA-EGTA.

    Quantitative RT-PCR:

    Article Title: Single-molecule imaging of transcription at damaged chromatin
    Article Snippet: One-half was digested with 10 U of MNase (Fermentas) for 30 min at 37°C before incubation with stop buffer [100 mM EDTA and proteinase K (20 μg/ml)] for 30 min at 55°C, and another half was treated with buffer alone (nondigested). .. Mononucleosome-sized DNA obtained after 30-min digestion and nondigested DNA were used after standard phenol-chloroform purification as templates for RT-qPCR.

    Incubation:

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells
    Article Snippet: Peak fractions containing all four subunits of the DKC1 complex, as determined by western blotting, were pooled and incubated with anti-FLAG (M2) agarose (Sigma Aldrich) for 3–4 hr, washed at 0.5 M NaCl HEGN and re-equilibrated with micrococcal nuclease (MNase) digestion buffer (25 mM Tris–HCl, pH 7.9, 20 mM NaCl, 60 mM KCl, 2 mM CaCl2 , 0.01% NP-40, 10% glycerol). .. Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Article Title: Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans
    Article Snippet: NETs from 10 wells were digested with 5 U/ml MNase (Fermentas), a non-processive nuclease that cuts DNA at linker sites. .. NETs from 10 wells were digested with 5 U/ml MNase (Fermentas), a non-processive nuclease that cuts DNA at linker sites.

    Article Title: A core viral protein binds host nucleosomes to sequester immune danger signals
    Article Snippet: Recombinant protein VII-His was then combined with nucleosomes at various molar ratios, incubated at room temperature for 15 minutes, and analyzed by native gel electrophoresis. .. Complexes were also digested with MNase (Affymetrix) by addition of 1 unit per µg of DNA for 147bp nucleosome experiments and 0.1 unit per µg of DNA for 195bp nucleosome experiments, incubated at 22°C for varying amounts of time followed by the addition of EGTA and guanidine thiocyanate to stop the reaction. .. The DNA fragments were then purified using a MinElute PCR purification kit (Qiagen) and analyzed on an Agilent 2100 Bioanalyzer as previously described .

    Article Title: Chromatin Structure and Transcription of the R1- and R2-Inserted rRNA Genes of Drosophila melanogaster
    Article Snippet: The reaction was stopped by adding EDTA and sodium dodecyl sulfate (SDS) to final concentrations of 10 mM and 0.02%, respectively. .. For micrococcal nuclease (MNase) digestion, the nucleus solutions were made 3 mM CaCl2 and 0.5 units/μl MNase (Fermentas) and incubated at 24°C for the specified time. .. Reactions were stopped as in the DNase I digestions.

    Article Title: Low-input and multiplexed microfluidic assay reveals epigenomic variation across cerebellum and prefrontal cortex
    Article Snippet: Chromatin fragmentation PIC (0.1 μl) and PMSF (0.1 μl of 100 mM) were freshly added in 10 μl of suspension (containing cells/nuclei ranging from 30 to 10,000). .. The sample was then mixed with 10 μl of 2× lysis buffer [4% Triton X-100, 100 mM tris (pH 7.5), 100 mM NaCl, and 30 mM MgCl2 ] and incubated at room temperature for 10 min. CaCl2 (1 μl of 0.1 M) and MNase (2.5 μl of 10 U/μl; 88216, Thermo Fisher Scientific) were rapidly mixed with the sample and incubated at room temperature for 10 min. EDTA (2.22 μl of 0.5 M) (pH 8) was added and incubated on ice for 10 min. .. The supernatant (~24 μl) was transferred to a new microcentrifuge tube and stored on ice.

    Article Title: DNA-encoded nucleosome occupancy is associated with transcription levels in the human malaria parasite Plasmodium falciparum
    Article Snippet: Nuclei were permeabilized by mild sonication for 4 cycles of 15 s ON and 45 s OFF at high intensity using a Bioruptor UCD-200. .. Next, 50 U MNase (USB corporation, Cleveland, OH) was added, followed by incubation for 5 min at 37°C. .. MNase was inactivated by the addition of EDTA to a final concentration of 5 mM and incubation for 5 min at RT with agitation.

    Article Title: Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity
    Article Snippet: The supernatant was discarded and the nuclei-containing-pellet washed twice in lysis buffer without detergent. .. Samples were incubated with 1U of MNase (Thermo Scientific) for 0, 5, 25 minutes at 37 °C, and the reaction was stopped with 2 mM EDTA-EGTA. .. Then, 10 μl of proteinase K (20 mg/ml) was added and incubated at 56 °C for 3 hs.

    Article Title: Single-molecule imaging of transcription at damaged chromatin
    Article Snippet: Nuclei were washed with digestion buffer [10 mM tris (pH 7.4), 15 mM NaCl, 60 mM KCl, and 1 mM CaCl2 ], centrifuged (3 min, 7000 rpm at 4°C), and resuspended in digestion buffer. .. One-half was digested with 10 U of MNase (Fermentas) for 30 min at 37°C before incubation with stop buffer [100 mM EDTA and proteinase K (20 μg/ml)] for 30 min at 55°C, and another half was treated with buffer alone (nondigested). .. Mononucleosome-sized DNA obtained after 30-min digestion and nondigested DNA were used after standard phenol-chloroform purification as templates for RT-qPCR.

    Article Title: A variant NuRD complex containing PWWP2A/B excludes MBD2/3 to regulate transcription at active genes
    Article Snippet: For each IP, 100 µl of chromatin was diluted with ChIP dilution buffer (1% Triton X-100, 1 mM EDTA, 20 mM Tris-HCl, pH 8, 150 mM NaCl) prior to pre-clearing with protein A agarose beads or magnetic Dynabeads (Invitrogen) blocked with 1 mg/mL BSA and 1 mg/mL yeast tRNA at 4 °C for 1 h. Precleared chromatin samples were further incubated overnight with relevant antibodies at 4 °C with rotation. .. The nuclei were resuspended in 1 ml of RSB + 0.25 M sucrose + 3 mM CaCl2 , treated with 200U of MNase (Fermentas) for 5 min at 37 °C, quenched with 4 µl of 1 M EDTA, then centrifuged at 2500 × g for 5 min.

    Article Title: The Arabidopsis SWI/SNF protein BAF60 mediates seedling growth control by modulating DNA accessibility
    Article Snippet: Five grams of 14-day-old seedlings were ground and nuclei were isolated with 4 °C buffer (0.25 M sucrose, 10 mM Tris-HCl, 10 mM MgCl2 , 1% Triton, 5 mM β-mercaptoethanol) containing proteinase inhibitor cocktail (Roche), filtered with a 63 μm filter, and incubated in MN buffer (20 mM Tris-HCl, 70 mM NaCl, 20 mM KCl, 5 mM MgCl2 , 3 mM CaCl2 ). .. MNase (100 U; Invitrogen®) was added to initiate the kinetics.

    Article Title: A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions
    Article Snippet: The cultures were further incubated on a rotary shaker for 2 h and mycelia was harvested by filtering, grind to fine powder under liquid nitrogen and lyophilized overnight. .. Approximately 20 mg of these lyophilized samples were suspended in 1 ml of MNase buffer (250 mM sucrose, 60 mM KCl, 15 mM NaCl, 0.5 mM CaCl2 , 3 mM MgCl2 ) and 300 µl of these suspensions were treated for 5 min with 0.5, 1 and 3 U of MNase (USB Corp., Cleveland, OH, USA) at 37°C.

    Activity Assay:

    Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns
    Article Snippet: Cells were cultured in six-well plates for > 24 h to 80% confluency and treated with various cytotoxic reagents for the indicated time periods. .. Released DNA from dying cells were digested with 500 mU/ml MNase (Thermo Scientific) for 5 min. Nuclease activity was stopped with 5 mM EDTA and the culture supernatants were collected and stored at −20 °C until quantification. .. Total genomic DNA from HeLa cells was extracted with 500 μ l DNazol (Molecular Research Center, Cincinnati, OH, USA).

    Expressing:

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells
    Article Snippet: Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr. .. Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Modification:

    Article Title: Locus-specific editing of histone modifications at endogenous enhancers using programmable TALE-LSD1 fusions
    Article Snippet: Quantitative measurements of histone modification levels were preformed in parallel using native ChIP. .. 0.01 U of MNase (ThermoScientific) was added to 1 ml lysis buffer (50mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM CaCl2) with EDTA free proteinase inhibitor.

    Western Blot:

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells
    Article Snippet: Peak fractions containing all four subunits of the DKC1 complex, as determined by western blotting, were pooled and incubated with anti-FLAG (M2) agarose (Sigma Aldrich) for 3–4 hr, washed at 0.5 M NaCl HEGN and re-equilibrated with micrococcal nuclease (MNase) digestion buffer (25 mM Tris–HCl, pH 7.9, 20 mM NaCl, 60 mM KCl, 2 mM CaCl2 , 0.01% NP-40, 10% glycerol). .. Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Transformation Assay:

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells
    Article Snippet: Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr. .. Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Derivative Assay:

    Article Title: A c-Myb mutant causes deregulated differentiation due to impaired histone binding and abrogated pioneer factor function
    Article Snippet: This results in a biotinylated 12-mer of 2437 bp and non-biotinylated 1622, 896 and 135 bp fragments derived from the pUC18 vector serving as competitor DNA in the nucleosome assembly reaction. .. MNase and immobilization to paramagnetic streptavidin beads (M280, Invitrogen) was performed as described in ( ).

    Flow Cytometry:

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells
    Article Snippet: Peak fractions were loaded immediately to a gravity column containing Heparin Sepharose 6 Fast Flow (GE Healthcare) pre-equilibrated with 0.3 M NaCl HEGN (25 mM HEPES, pH 7.9, 0.1 mM EDTA, 10% glycerol, 0.02% NP-40). .. Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Gas Chromatography:

    Article Title: Permissiveness of human hepatoma cell lines for HCV infection
    Article Snippet: Extracellular RNA from cell supernatants or intracellular HCV RNA from lysed cell pellets was treated with 4.5 units of S7 Micrococcal nuclease (Fermentas, Glen Burnie, MD) for 30 min at room temperature, and nuclease-treated RNA was isolated by the guanidine thiocyanate (GTC) method using 1.6 × GTC containing 2 μg of murine total liver RNA and following standard protocols [ ]. .. HCV, human GAPDH, murine GAPDH, human CD81, human SR-BI, human CLDN1, human OCLN and human ISG56 RNA levels were determined relative to standard curves comprised of serial dilutions of plasmids containing the JFH-1 HCV cDNA or the human GAPDH, murine GAPDH, human CD81, human SR-BI, human CLDN1, human OCLN and human ISG56 coding sequences, respectively.

    Southern Blot:

    Article Title: Chromatin Structure and Transcription of the R1- and R2-Inserted rRNA Genes of Drosophila melanogaster
    Article Snippet: Paragraph title: Nuclease digestions and Southern blotting. ... For micrococcal nuclease (MNase) digestion, the nucleus solutions were made 3 mM CaCl2 and 0.5 units/μl MNase (Fermentas) and incubated at 24°C for the specified time.

    Immunoprecipitation:

    Article Title: DNA-encoded nucleosome occupancy is associated with transcription levels in the human malaria parasite Plasmodium falciparum
    Article Snippet: Next, 50 U MNase (USB corporation, Cleveland, OH) was added, followed by incubation for 5 min at 37°C. .. MNase was inactivated by the addition of EDTA to a final concentration of 5 mM and incubation for 5 min at RT with agitation.

    Article Title: ATRX binds to atypical chromatin domains at the 3′ exons of zinc finger genes to preserve H3K9me3 enrichment
    Article Snippet: Native ChIP was performed as previously described with minor changes. .. Briefly, nuclei isolated from K562 and LAN6 (4–6 × 106 ) cells were treated with MNase (Affymetrix Cat #70196Y) and ∼40–80 μg of digested chromatin was immunoprecipitated with specific antibodies. .. The immunoprecipitated material was treated with Proteinase K for 3 h at 56°C and purified using the Qiagen MinElute PCR purification kit.

    Protease Inhibitor:

    Article Title: DNA-encoded nucleosome occupancy is associated with transcription levels in the human malaria parasite Plasmodium falciparum
    Article Snippet: Parasite nuclei were resuspended in MNase buffer (50 mM Tris-HCl pH 7.4, 4 mM MgCl2 , 1 mM CaCl2 , 2 mM AEBSF, and EDTA-free protease inhibitor cocktail) and transferred to 1.5 ml TPX polymethylpentene tubes. .. Next, 50 U MNase (USB corporation, Cleveland, OH) was added, followed by incubation for 5 min at 37°C.

    Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene
    Article Snippet: Pelleted nuclei were resuspended in 200 μL buffer A (0.34 M sucrose, 15 mM HEPES pH 7.4, 15 mM NaCl, 60 mM KCl, 4 mM MgCl2 , 1 mM DTT, 0.1 mM PMSF, 1:1000 protease inhibitor cocktail) and further diluted to 600 ng/μL. .. MNase (Affymetrix) digestion reactions were carried out on 100 μg or more chromatin using 0.05–0.2 U/μg chromatin in buffer A supplemented with 3 mM CaCl2 , 1 mM DTT, 0.1 mM PMSF and 1:1000 protease inhibitor cocktail, for 10 min at 37°C. .. The reaction was quenched with 5 mM EGTA on ice and centrifuged at 13,500×g for 10 min at 4°C.

    Cell Culture:

    Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns
    Article Snippet: Cells were cultured in six-well plates for > 24 h to 80% confluency and treated with various cytotoxic reagents for the indicated time periods. .. Released DNA from dying cells were digested with 500 mU/ml MNase (Thermo Scientific) for 5 min. Nuclease activity was stopped with 5 mM EDTA and the culture supernatants were collected and stored at −20 °C until quantification.

    other:

    Article Title: Low-input and multiplexed microfluidic assay reveals epigenomic variation across cerebellum and prefrontal cortex
    Article Snippet: Chromatin fragmentation PIC (0.1 μl) and PMSF (0.1 μl of 100 mM) were freshly added in 10 μl of suspension (containing cells/nuclei ranging from 30 to 10,000).

    Polymerase Chain Reaction:

    Article Title: Permissiveness of human hepatoma cell lines for HCV infection
    Article Snippet: Extracellular RNA from cell supernatants or intracellular HCV RNA from lysed cell pellets was treated with 4.5 units of S7 Micrococcal nuclease (Fermentas, Glen Burnie, MD) for 30 min at room temperature, and nuclease-treated RNA was isolated by the guanidine thiocyanate (GTC) method using 1.6 × GTC containing 2 μg of murine total liver RNA and following standard protocols [ ]. .. HCV, human GAPDH, murine GAPDH, human CD81, human SR-BI, human CLDN1, human OCLN and human ISG56 RNA levels were determined relative to standard curves comprised of serial dilutions of plasmids containing the JFH-1 HCV cDNA or the human GAPDH, murine GAPDH, human CD81, human SR-BI, human CLDN1, human OCLN and human ISG56 coding sequences, respectively.

    Sonication:

    Article Title: DNA-encoded nucleosome occupancy is associated with transcription levels in the human malaria parasite Plasmodium falciparum
    Article Snippet: Nuclei were permeabilized by mild sonication for 4 cycles of 15 s ON and 45 s OFF at high intensity using a Bioruptor UCD-200. .. Next, 50 U MNase (USB corporation, Cleveland, OH) was added, followed by incubation for 5 min at 37°C.

    Binding Assay:

    Article Title: A core viral protein binds host nucleosomes to sequester immune danger signals
    Article Snippet: Paragraph title: Nucleosome in vitro binding and MNase digestion assays ... Complexes were also digested with MNase (Affymetrix) by addition of 1 unit per µg of DNA for 147bp nucleosome experiments and 0.1 unit per µg of DNA for 195bp nucleosome experiments, incubated at 22°C for varying amounts of time followed by the addition of EGTA and guanidine thiocyanate to stop the reaction.

    Article Title: Locus-specific editing of histone modifications at endogenous enhancers using programmable TALE-LSD1 fusions
    Article Snippet: 0.01 U of MNase (ThermoScientific) was added to 1 ml lysis buffer (50mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM CaCl2) with EDTA free proteinase inhibitor. .. The lysed sample was split into 96 well plate format for ChIP with H3K4me2 (abcam ab32356), H3K27ac (Active Motif 39133), H3K4me3 (Abcam ab8580), or H3K4me1 (Abcam ab8895).

    Article Title: A c-Myb mutant causes deregulated differentiation due to impaired histone binding and abrogated pioneer factor function
    Article Snippet: Paragraph title: In vitro nucleosome binding assay ... MNase and immobilization to paramagnetic streptavidin beads (M280, Invitrogen) was performed as described in ( ).

    Cellular Antioxidant Activity Assay:

    Article Title: Permissiveness of human hepatoma cell lines for HCV infection
    Article Snippet: Extracellular RNA from cell supernatants or intracellular HCV RNA from lysed cell pellets was treated with 4.5 units of S7 Micrococcal nuclease (Fermentas, Glen Burnie, MD) for 30 min at room temperature, and nuclease-treated RNA was isolated by the guanidine thiocyanate (GTC) method using 1.6 × GTC containing 2 μg of murine total liver RNA and following standard protocols [ ]. .. HCV, human GAPDH, murine GAPDH, human CD81, human SR-BI, human CLDN1, human OCLN and human ISG56 RNA levels were determined relative to standard curves comprised of serial dilutions of plasmids containing the JFH-1 HCV cDNA or the human GAPDH, murine GAPDH, human CD81, human SR-BI, human CLDN1, human OCLN and human ISG56 coding sequences, respectively.

    ChIP-sequencing:

    Article Title: A variant NuRD complex containing PWWP2A/B excludes MBD2/3 to regulate transcription at active genes
    Article Snippet: For calibrated ChIP-seq of Pol II Ser5P and Pol II Ser2P, 40 million ES cells and 10 million Drosophila SG4 cells were pooled in PBS prior to cross-linking. .. The nuclei were resuspended in 1 ml of RSB + 0.25 M sucrose + 3 mM CaCl2 , treated with 200U of MNase (Fermentas) for 5 min at 37 °C, quenched with 4 µl of 1 M EDTA, then centrifuged at 2500 × g for 5 min.

    Article Title: The Arabidopsis SWI/SNF protein BAF60 mediates seedling growth control by modulating DNA accessibility
    Article Snippet: MNase (100 U; Invitrogen®) was added to initiate the kinetics. .. Purified DNA was run on a 1% agarose gel and the band corresponding to mononucleosome was excised and purified with a MinElute Gel Extraction Kit (Qiagen).

    Nucleic Acid Electrophoresis:

    Article Title: A core viral protein binds host nucleosomes to sequester immune danger signals
    Article Snippet: Recombinant protein VII-His was then combined with nucleosomes at various molar ratios, incubated at room temperature for 15 minutes, and analyzed by native gel electrophoresis. .. Complexes were also digested with MNase (Affymetrix) by addition of 1 unit per µg of DNA for 147bp nucleosome experiments and 0.1 unit per µg of DNA for 195bp nucleosome experiments, incubated at 22°C for varying amounts of time followed by the addition of EGTA and guanidine thiocyanate to stop the reaction.

    Article Title: A c-Myb mutant causes deregulated differentiation due to impaired histone binding and abrogated pioneer factor function
    Article Snippet: To determine the point of saturation, the 12-mer 601 array including competitor DNA was reconstituted with increasing concentrations of recombinant histone octamers and assayed by native gel electrophoresis to reach the saturation point ( ). .. MNase and immobilization to paramagnetic streptavidin beads (M280, Invitrogen) was performed as described in ( ).

    Sensitive Assay:

    Article Title: The Arabidopsis SWI/SNF protein BAF60 mediates seedling growth control by modulating DNA accessibility
    Article Snippet: Paragraph title: Microccocal nuclease sensitivity assay ... MNase (100 U; Invitrogen®) was added to initiate the kinetics.

    Magnetic Beads:

    Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene
    Article Snippet: MNase (Affymetrix) digestion reactions were carried out on 100 μg or more chromatin using 0.05–0.2 U/μg chromatin in buffer A supplemented with 3 mM CaCl2 , 1 mM DTT, 0.1 mM PMSF and 1:1000 protease inhibitor cocktail, for 10 min at 37°C. .. MNase (Affymetrix) digestion reactions were carried out on 100 μg or more chromatin using 0.05–0.2 U/μg chromatin in buffer A supplemented with 3 mM CaCl2 , 1 mM DTT, 0.1 mM PMSF and 1:1000 protease inhibitor cocktail, for 10 min at 37°C.

    Isolation:

    Article Title: Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans
    Article Snippet: We isolated NET proteins as described in ‘Purification of NET-proteins’. .. NETs from 10 wells were digested with 5 U/ml MNase (Fermentas), a non-processive nuclease that cuts DNA at linker sites.

    Article Title: Permissiveness of human hepatoma cell lines for HCV infection
    Article Snippet: Total intracellular RNA was isolated in 1 × Nucleic Acid Purification Lysis Solution (Applied Biosystems, Foster City, CA) and purified using an ABI PRISM™ 6100 Nucleic Acid PrepStation (Applied Biosystems), as per the manufacturer's instructions. .. Extracellular RNA from cell supernatants or intracellular HCV RNA from lysed cell pellets was treated with 4.5 units of S7 Micrococcal nuclease (Fermentas, Glen Burnie, MD) for 30 min at room temperature, and nuclease-treated RNA was isolated by the guanidine thiocyanate (GTC) method using 1.6 × GTC containing 2 μg of murine total liver RNA and following standard protocols [ ]. .. One μg of purified RNA was used for cDNA synthesis using the TaqMan reverse transcription reagents (Applied Biosystems), followed by SYBR green RTqPCR using an Applied Biosystems 7300 real-time thermocycler (Applied Biosystems).

    Article Title: A variant NuRD complex containing PWWP2A/B excludes MBD2/3 to regulate transcription at active genes
    Article Snippet: Antibody-bound chromatin was isolated on blocked protein A beads, and followed by washes with low salt buffer (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl), high salt buffer ((0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)), and twice with TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) buffer. .. The nuclei were resuspended in 1 ml of RSB + 0.25 M sucrose + 3 mM CaCl2 , treated with 200U of MNase (Fermentas) for 5 min at 37 °C, quenched with 4 µl of 1 M EDTA, then centrifuged at 2500 × g for 5 min.

    Article Title: The Arabidopsis SWI/SNF protein BAF60 mediates seedling growth control by modulating DNA accessibility
    Article Snippet: Five grams of 14-day-old seedlings were ground and nuclei were isolated with 4 °C buffer (0.25 M sucrose, 10 mM Tris-HCl, 10 mM MgCl2 , 1% Triton, 5 mM β-mercaptoethanol) containing proteinase inhibitor cocktail (Roche), filtered with a 63 μm filter, and incubated in MN buffer (20 mM Tris-HCl, 70 mM NaCl, 20 mM KCl, 5 mM MgCl2 , 3 mM CaCl2 ). .. MNase (100 U; Invitrogen®) was added to initiate the kinetics.

    Article Title: ATRX binds to atypical chromatin domains at the 3′ exons of zinc finger genes to preserve H3K9me3 enrichment
    Article Snippet: Native ChIP was performed as previously described with minor changes. .. Briefly, nuclei isolated from K562 and LAN6 (4–6 × 106 ) cells were treated with MNase (Affymetrix Cat #70196Y) and ∼40–80 μg of digested chromatin was immunoprecipitated with specific antibodies. .. The immunoprecipitated material was treated with Proteinase K for 3 h at 56°C and purified using the Qiagen MinElute PCR purification kit.

    Lysis:

    Article Title: Permissiveness of human hepatoma cell lines for HCV infection
    Article Snippet: Total intracellular RNA was isolated in 1 × Nucleic Acid Purification Lysis Solution (Applied Biosystems, Foster City, CA) and purified using an ABI PRISM™ 6100 Nucleic Acid PrepStation (Applied Biosystems), as per the manufacturer's instructions. .. Extracellular RNA from cell supernatants or intracellular HCV RNA from lysed cell pellets was treated with 4.5 units of S7 Micrococcal nuclease (Fermentas, Glen Burnie, MD) for 30 min at room temperature, and nuclease-treated RNA was isolated by the guanidine thiocyanate (GTC) method using 1.6 × GTC containing 2 μg of murine total liver RNA and following standard protocols [ ].

    Article Title: Locus-specific editing of histone modifications at endogenous enhancers using programmable TALE-LSD1 fusions
    Article Snippet: Quantitative measurements of histone modification levels were preformed in parallel using native ChIP. .. 0.01 U of MNase (ThermoScientific) was added to 1 ml lysis buffer (50mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM CaCl2) with EDTA free proteinase inhibitor. .. For each transfected sample, 260 ul of MNase:Lysis buffer was added and incubated for 15 minutes at 25 C, and 20 minutes at 37C.

    Article Title: Low-input and multiplexed microfluidic assay reveals epigenomic variation across cerebellum and prefrontal cortex
    Article Snippet: Chromatin fragmentation PIC (0.1 μl) and PMSF (0.1 μl of 100 mM) were freshly added in 10 μl of suspension (containing cells/nuclei ranging from 30 to 10,000). .. The sample was then mixed with 10 μl of 2× lysis buffer [4% Triton X-100, 100 mM tris (pH 7.5), 100 mM NaCl, and 30 mM MgCl2 ] and incubated at room temperature for 10 min. CaCl2 (1 μl of 0.1 M) and MNase (2.5 μl of 10 U/μl; 88216, Thermo Fisher Scientific) were rapidly mixed with the sample and incubated at room temperature for 10 min. EDTA (2.22 μl of 0.5 M) (pH 8) was added and incubated on ice for 10 min. .. The supernatant (~24 μl) was transferred to a new microcentrifuge tube and stored on ice.

    Article Title: Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity
    Article Snippet: The supernatant was discarded and the nuclei-containing-pellet washed twice in lysis buffer without detergent. .. Samples were incubated with 1U of MNase (Thermo Scientific) for 0, 5, 25 minutes at 37 °C, and the reaction was stopped with 2 mM EDTA-EGTA.

    Electrophoretic Mobility Shift Assay:

    Article Title: A core viral protein binds host nucleosomes to sequester immune danger signals
    Article Snippet: Gel shift and MNase digestion assays were carried out as previously described , , . .. Complexes were also digested with MNase (Affymetrix) by addition of 1 unit per µg of DNA for 147bp nucleosome experiments and 0.1 unit per µg of DNA for 195bp nucleosome experiments, incubated at 22°C for varying amounts of time followed by the addition of EGTA and guanidine thiocyanate to stop the reaction.

    Purification:

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells
    Article Snippet: Paragraph title: Reconstitution and purification of the DKC1 complexes ... Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Article Title: Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans
    Article Snippet: We isolated NET proteins as described in ‘Purification of NET-proteins’. .. NETs from 10 wells were digested with 5 U/ml MNase (Fermentas), a non-processive nuclease that cuts DNA at linker sites.

    Article Title: Permissiveness of human hepatoma cell lines for HCV infection
    Article Snippet: Total intracellular RNA was isolated in 1 × Nucleic Acid Purification Lysis Solution (Applied Biosystems, Foster City, CA) and purified using an ABI PRISM™ 6100 Nucleic Acid PrepStation (Applied Biosystems), as per the manufacturer's instructions. .. Extracellular RNA from cell supernatants or intracellular HCV RNA from lysed cell pellets was treated with 4.5 units of S7 Micrococcal nuclease (Fermentas, Glen Burnie, MD) for 30 min at room temperature, and nuclease-treated RNA was isolated by the guanidine thiocyanate (GTC) method using 1.6 × GTC containing 2 μg of murine total liver RNA and following standard protocols [ ].

    Article Title: A core viral protein binds host nucleosomes to sequester immune danger signals
    Article Snippet: Briefly, nucleosomes were reconstituted by incubating purified recombinant histones with ‘601’ DNA of either 195 or 147bp over a series of dialysis. .. Complexes were also digested with MNase (Affymetrix) by addition of 1 unit per µg of DNA for 147bp nucleosome experiments and 0.1 unit per µg of DNA for 195bp nucleosome experiments, incubated at 22°C for varying amounts of time followed by the addition of EGTA and guanidine thiocyanate to stop the reaction.

    Article Title: Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity
    Article Snippet: Samples were incubated with 1U of MNase (Thermo Scientific) for 0, 5, 25 minutes at 37 °C, and the reaction was stopped with 2 mM EDTA-EGTA. .. Samples were incubated with 1U of MNase (Thermo Scientific) for 0, 5, 25 minutes at 37 °C, and the reaction was stopped with 2 mM EDTA-EGTA.

    Article Title: A variant NuRD complex containing PWWP2A/B excludes MBD2/3 to regulate transcription at active genes
    Article Snippet: The DNA was eluted with 1% SDS and 100 mM NaHCO3 , and reversed cross-linked at 65 °C overnight in the presence of 200 mM NaCl, following by treatment with RNaseA and Proteinase K. DNA was purified by ChIP DNA Clean and Concentrator kit (Zymo) and quantified by dsDNA Qubit reagents. .. The nuclei were resuspended in 1 ml of RSB + 0.25 M sucrose + 3 mM CaCl2 , treated with 200U of MNase (Fermentas) for 5 min at 37 °C, quenched with 4 µl of 1 M EDTA, then centrifuged at 2500 × g for 5 min.

    Article Title: The Arabidopsis SWI/SNF protein BAF60 mediates seedling growth control by modulating DNA accessibility
    Article Snippet: MNase (100 U; Invitrogen®) was added to initiate the kinetics. .. MNase (100 U; Invitrogen®) was added to initiate the kinetics.

    Article Title: A c-Myb mutant causes deregulated differentiation due to impaired histone binding and abrogated pioneer factor function
    Article Snippet: A 12 bp PstI-XbaI fragment was purified away over the Quick Spin Sephadex G-50 column. .. MNase and immobilization to paramagnetic streptavidin beads (M280, Invitrogen) was performed as described in ( ).

    Sequencing:

    Article Title: Noncoding transcription influences the replication initiation program through chromatin regulation
    Article Snippet: MNase (Thermo Fisher Scientific) treatment was performed at a previously optimized concentration to have comparable intensity of both mono- and di-nucleosomes within and between the samples. .. MNase (Thermo Fisher Scientific) treatment was performed at a previously optimized concentration to have comparable intensity of both mono- and di-nucleosomes within and between the samples.

    Staining:

    Article Title: Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity
    Article Snippet: Samples were incubated with 1U of MNase (Thermo Scientific) for 0, 5, 25 minutes at 37 °C, and the reaction was stopped with 2 mM EDTA-EGTA. .. Samples were incubated with 1U of MNase (Thermo Scientific) for 0, 5, 25 minutes at 37 °C, and the reaction was stopped with 2 mM EDTA-EGTA.

    Article Title: A c-Myb mutant causes deregulated differentiation due to impaired histone binding and abrogated pioneer factor function
    Article Snippet: MNase and immobilization to paramagnetic streptavidin beads (M280, Invitrogen) was performed as described in ( ). .. For pulldown experiments, beads alone, biotinylated 12-mer 601 arrays or biotinlyated chromatinized 12-mer 601 arrays were immobilized to 20 μl streptdavidin beads and incubated on a rotating wheel with 1 μg of recombinant GST, GST-NR123 or GST-NR123D152V in EX100 (10 mM HEPES [pH 7.6], 100 mM NaCl, 1.5 mM MgCl2 , 0.5 mM EGTA, 10% [vol/vol] glycerol, 0.2 mM PMSF, 1 mM DTT) containing 0.01% (vol/vol) NP-40 and 20 μg bovine serum albumin, for 1 h at 4°C.

    Activated Clotting Time Assay:

    Article Title: Permissiveness of human hepatoma cell lines for HCV infection
    Article Snippet: Extracellular RNA from cell supernatants or intracellular HCV RNA from lysed cell pellets was treated with 4.5 units of S7 Micrococcal nuclease (Fermentas, Glen Burnie, MD) for 30 min at room temperature, and nuclease-treated RNA was isolated by the guanidine thiocyanate (GTC) method using 1.6 × GTC containing 2 μg of murine total liver RNA and following standard protocols [ ]. .. HCV, human GAPDH, murine GAPDH, human CD81, human SR-BI, human CLDN1, human OCLN and human ISG56 RNA levels were determined relative to standard curves comprised of serial dilutions of plasmids containing the JFH-1 HCV cDNA or the human GAPDH, murine GAPDH, human CD81, human SR-BI, human CLDN1, human OCLN and human ISG56 coding sequences, respectively.

    Chromatin Immunoprecipitation:

    Article Title: Locus-specific editing of histone modifications at endogenous enhancers using programmable TALE-LSD1 fusions
    Article Snippet: Paragraph title: Native ChIP ... 0.01 U of MNase (ThermoScientific) was added to 1 ml lysis buffer (50mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM CaCl2) with EDTA free proteinase inhibitor.

    Article Title: Restitution of gene expression and histone acetylation signatures altered by hepatitis B virus through antiviral microRNA-like molecules in nontransformed murine hepatocytes
    Article Snippet: MNase digest time courses of each 1 × 106 cells were performed for 0, 1, 2, 4, 8, 16 or 20 minutes with 0.15 U/μL MNase (Thermo Fisher Scientific, Waltham, MA, USA). .. The reactions were stopped by directly mixing 50 μL phenol:chloroform:isoamylalcohol with each sample followed by DNA extraction.

    Article Title: Single-molecule imaging of transcription at damaged chromatin
    Article Snippet: Briefly, EX2 cells were prepared as described for the ChIP protocol and, after TA incubation, collected by trypsinization. .. One-half was digested with 10 U of MNase (Fermentas) for 30 min at 37°C before incubation with stop buffer [100 mM EDTA and proteinase K (20 μg/ml)] for 30 min at 55°C, and another half was treated with buffer alone (nondigested).

    Article Title: A variant NuRD complex containing PWWP2A/B excludes MBD2/3 to regulate transcription at active genes
    Article Snippet: Paragraph title: Chromatin immunoprecipitation ... The nuclei were resuspended in 1 ml of RSB + 0.25 M sucrose + 3 mM CaCl2 , treated with 200U of MNase (Fermentas) for 5 min at 37 °C, quenched with 4 µl of 1 M EDTA, then centrifuged at 2500 × g for 5 min.

    Article Title: ATRX binds to atypical chromatin domains at the 3′ exons of zinc finger genes to preserve H3K9me3 enrichment
    Article Snippet: Paragraph title: Native ChIP ... Briefly, nuclei isolated from K562 and LAN6 (4–6 × 106 ) cells were treated with MNase (Affymetrix Cat #70196Y) and ∼40–80 μg of digested chromatin was immunoprecipitated with specific antibodies.

    Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene
    Article Snippet: Paragraph title: Native and crosslinked ChIP and next-generation sequencing ... MNase (Affymetrix) digestion reactions were carried out on 100 μg or more chromatin using 0.05–0.2 U/μg chromatin in buffer A supplemented with 3 mM CaCl2 , 1 mM DTT, 0.1 mM PMSF and 1:1000 protease inhibitor cocktail, for 10 min at 37°C.

    Plasmid Preparation:

    Article Title: A c-Myb mutant causes deregulated differentiation due to impaired histone binding and abrogated pioneer factor function
    Article Snippet: This results in a biotinylated 12-mer of 2437 bp and non-biotinylated 1622, 896 and 135 bp fragments derived from the pUC18 vector serving as competitor DNA in the nucleosome assembly reaction. .. MNase and immobilization to paramagnetic streptavidin beads (M280, Invitrogen) was performed as described in ( ).

    Recombinant:

    Article Title: A core viral protein binds host nucleosomes to sequester immune danger signals
    Article Snippet: Recombinant protein VII-His was then combined with nucleosomes at various molar ratios, incubated at room temperature for 15 minutes, and analyzed by native gel electrophoresis. .. Complexes were also digested with MNase (Affymetrix) by addition of 1 unit per µg of DNA for 147bp nucleosome experiments and 0.1 unit per µg of DNA for 195bp nucleosome experiments, incubated at 22°C for varying amounts of time followed by the addition of EGTA and guanidine thiocyanate to stop the reaction.

    Article Title: A c-Myb mutant causes deregulated differentiation due to impaired histone binding and abrogated pioneer factor function
    Article Snippet: To determine the point of saturation, the 12-mer 601 array including competitor DNA was reconstituted with increasing concentrations of recombinant histone octamers and assayed by native gel electrophoresis to reach the saturation point ( ). .. MNase and immobilization to paramagnetic streptavidin beads (M280, Invitrogen) was performed as described in ( ).

    Agarose Gel Electrophoresis:

    Article Title: Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity
    Article Snippet: Samples were incubated with 1U of MNase (Thermo Scientific) for 0, 5, 25 minutes at 37 °C, and the reaction was stopped with 2 mM EDTA-EGTA. .. Samples were incubated with 1U of MNase (Thermo Scientific) for 0, 5, 25 minutes at 37 °C, and the reaction was stopped with 2 mM EDTA-EGTA.

    Article Title: The Arabidopsis SWI/SNF protein BAF60 mediates seedling growth control by modulating DNA accessibility
    Article Snippet: MNase (100 U; Invitrogen®) was added to initiate the kinetics. .. MNase (100 U; Invitrogen®) was added to initiate the kinetics.

    In Vitro:

    Article Title: A core viral protein binds host nucleosomes to sequester immune danger signals
    Article Snippet: Paragraph title: Nucleosome in vitro binding and MNase digestion assays ... Complexes were also digested with MNase (Affymetrix) by addition of 1 unit per µg of DNA for 147bp nucleosome experiments and 0.1 unit per µg of DNA for 195bp nucleosome experiments, incubated at 22°C for varying amounts of time followed by the addition of EGTA and guanidine thiocyanate to stop the reaction.

    Article Title: A c-Myb mutant causes deregulated differentiation due to impaired histone binding and abrogated pioneer factor function
    Article Snippet: Paragraph title: In vitro nucleosome binding assay ... MNase and immobilization to paramagnetic streptavidin beads (M280, Invitrogen) was performed as described in ( ).

    Nucleic Acid Purification:

    Article Title: Permissiveness of human hepatoma cell lines for HCV infection
    Article Snippet: Total intracellular RNA was isolated in 1 × Nucleic Acid Purification Lysis Solution (Applied Biosystems, Foster City, CA) and purified using an ABI PRISM™ 6100 Nucleic Acid PrepStation (Applied Biosystems), as per the manufacturer's instructions. .. Extracellular RNA from cell supernatants or intracellular HCV RNA from lysed cell pellets was treated with 4.5 units of S7 Micrococcal nuclease (Fermentas, Glen Burnie, MD) for 30 min at room temperature, and nuclease-treated RNA was isolated by the guanidine thiocyanate (GTC) method using 1.6 × GTC containing 2 μg of murine total liver RNA and following standard protocols [ ].

    Next-Generation Sequencing:

    Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene
    Article Snippet: Paragraph title: Native and crosslinked ChIP and next-generation sequencing ... MNase (Affymetrix) digestion reactions were carried out on 100 μg or more chromatin using 0.05–0.2 U/μg chromatin in buffer A supplemented with 3 mM CaCl2 , 1 mM DTT, 0.1 mM PMSF and 1:1000 protease inhibitor cocktail, for 10 min at 37°C.

    Concentration Assay:

    Article Title: Noncoding transcription influences the replication initiation program through chromatin regulation
    Article Snippet: Chromatin was then collected by centrifugation and resuspended in NP-buffer (0.5 mM spermidine, 1 mM β-ME, 0.075% NP-40, 50 mM NaCl, 10 mM Tris at pH 7.4, 5 mM MgCl2 , 1 mM CaCl2 ). .. MNase (Thermo Fisher Scientific) treatment was performed at a previously optimized concentration to have comparable intensity of both mono- and di-nucleosomes within and between the samples. .. MNase treatment was followed by de-crosslinking and protease treatment, and DNA was extracted using NucleoSpin gel and PCR extraction columns (Macherey-Nagel).

    Ethanol Precipitation:

    Article Title: Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity
    Article Snippet: Samples were incubated with 1U of MNase (Thermo Scientific) for 0, 5, 25 minutes at 37 °C, and the reaction was stopped with 2 mM EDTA-EGTA. .. Samples were incubated with 1U of MNase (Thermo Scientific) for 0, 5, 25 minutes at 37 °C, and the reaction was stopped with 2 mM EDTA-EGTA.

    CTG Assay:

    Article Title: Permissiveness of human hepatoma cell lines for HCV infection
    Article Snippet: Extracellular RNA from cell supernatants or intracellular HCV RNA from lysed cell pellets was treated with 4.5 units of S7 Micrococcal nuclease (Fermentas, Glen Burnie, MD) for 30 min at room temperature, and nuclease-treated RNA was isolated by the guanidine thiocyanate (GTC) method using 1.6 × GTC containing 2 μg of murine total liver RNA and following standard protocols [ ]. .. HCV, human GAPDH, murine GAPDH, human CD81, human SR-BI, human CLDN1, human OCLN and human ISG56 RNA levels were determined relative to standard curves comprised of serial dilutions of plasmids containing the JFH-1 HCV cDNA or the human GAPDH, murine GAPDH, human CD81, human SR-BI, human CLDN1, human OCLN and human ISG56 coding sequences, respectively.

    Gel Extraction:

    Article Title: The Arabidopsis SWI/SNF protein BAF60 mediates seedling growth control by modulating DNA accessibility
    Article Snippet: MNase (100 U; Invitrogen®) was added to initiate the kinetics. .. MNase (100 U; Invitrogen®) was added to initiate the kinetics.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    Thermo Fisher mnase
    <t>DKC1-associated</t> RNAs in recombinant DKC1 complexes are resistant to extensive <t>MNase</t> digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010
    Mnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mnase/product/Thermo Fisher
    Average 95 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    mnase - by Bioz Stars, 2019-12
    95/100 stars
      Buy from Supplier

    Image Search Results


    DKC1-associated RNAs in recombinant DKC1 complexes are resistant to extensive MNase digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010

    Journal: eLife

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

    doi: 10.7554/eLife.03573

    Figure Lengend Snippet: DKC1-associated RNAs in recombinant DKC1 complexes are resistant to extensive MNase digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010

    Article Snippet: Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Techniques: Recombinant, Purification, Labeling, Stable Transfection

    Micrococcal nuclease (MNase)-treated recombinant DKC1 complexes remain structurally intact. Recombinant wild-type (WT) and various mutant DKC1 complexes are mock treated (−) or digested extensively with MNase (+), and washed extensively to remove any dissociated RNAs prior to FLAG peptide elution. Eluted protein complexes are analyzed by Coomassie Blue staining. DOI: http://dx.doi.org/10.7554/eLife.03573.009

    Journal: eLife

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

    doi: 10.7554/eLife.03573

    Figure Lengend Snippet: Micrococcal nuclease (MNase)-treated recombinant DKC1 complexes remain structurally intact. Recombinant wild-type (WT) and various mutant DKC1 complexes are mock treated (−) or digested extensively with MNase (+), and washed extensively to remove any dissociated RNAs prior to FLAG peptide elution. Eluted protein complexes are analyzed by Coomassie Blue staining. DOI: http://dx.doi.org/10.7554/eLife.03573.009

    Article Snippet: Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Techniques: Recombinant, Mutagenesis, Staining

    MNase digestion moderately increases DKC1 coactivator activity. Mock (−) or MNase-treated (+) WT and Nop10 R34W DKC1 complexes are assayed in in vitro transcription reactions (over a fourfold concentration range) supplemented with OCT4, SOX2, recombinant XPC complex and SCC-B. DOI: http://dx.doi.org/10.7554/eLife.03573.011

    Journal: eLife

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

    doi: 10.7554/eLife.03573

    Figure Lengend Snippet: MNase digestion moderately increases DKC1 coactivator activity. Mock (−) or MNase-treated (+) WT and Nop10 R34W DKC1 complexes are assayed in in vitro transcription reactions (over a fourfold concentration range) supplemented with OCT4, SOX2, recombinant XPC complex and SCC-B. DOI: http://dx.doi.org/10.7554/eLife.03573.011

    Article Snippet: Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Techniques: Activity Assay, In Vitro, Concentration Assay, Recombinant

    Release of nucleosomes and DAMPs from amino-acid-depleted HeLa cells. ( A ) An inverted microscopic image of HeLa cells in the condition of amino-acid depletion. Arrows designate dying HeLa cells. ( B ) Genomic sequences of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), Fas, cytochrome oxidase subunit 1 ( Co1 ) and ATP synthase subunit 6 ( ATP6 ) were PCR amplified from extracellularly released DNA, genomic or mitochondrial DNAs. ( C ) Inverted and fluorescent microscopic images were taken from amino-acid-deprived HeLa cells in the presence of SYTOX, a membrane-impermeable DNA dye. HeLa cells deprived of amino acids for 15 h were fluorescence stained with histone H1 or IL6 antibodies ( D ), histone H2A, H2B, H3 or H4 antibodies ( E ) or HMGB1, Hsp90 or ERp57 antibodies ( F ) in combination with histone H1 antibody and 4',6-diamidino-2-phenylindole (DAPI). ( G ) Amino-acid-deprived HeLa cells were stained with SYTOX to determine viability, fixed and stained with DAPI and histone H1 antibodies. ( H ) Amino-acid-deprived HeLa cells were untreated or treated with MNase (500 mU/ml) for 10 min. Released DNA was quantitated at the indicated times. Data from triplicate samples are presented as mean±S.D. ( I ) Conditioned media from amino-acid-deprived HeLa cells treated or untreated with MNase were western blotted with histone H1, 2B, H3, H4, IL6, ERp57, HMGB1 or Hsp90 antibodies. ( J ) Images captured every hour from live imaging of amino-acid-deprived HeLa cells with SYTOX (green) and DRAQ5, membrane-permeable DNA dye (red). ( K ) SYTOX fluorescent intensities were measured from circularized areas of live imaging of amino-acid-deprived cells in 5-min intervals. ( L ) TEM images of control cells ( L a) and amino-acid-deprived HeLa cells ( L b– L d). ( M ) Amino-acid-deprived HeLa cells were fluorescence stained with lamin and nuclear pore antibodies, or lamin antibody and wheat germ agglutinin (WGE)

    Journal: Cell Death & Disease

    Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns

    doi: 10.1038/cddis.2014.450

    Figure Lengend Snippet: Release of nucleosomes and DAMPs from amino-acid-depleted HeLa cells. ( A ) An inverted microscopic image of HeLa cells in the condition of amino-acid depletion. Arrows designate dying HeLa cells. ( B ) Genomic sequences of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), Fas, cytochrome oxidase subunit 1 ( Co1 ) and ATP synthase subunit 6 ( ATP6 ) were PCR amplified from extracellularly released DNA, genomic or mitochondrial DNAs. ( C ) Inverted and fluorescent microscopic images were taken from amino-acid-deprived HeLa cells in the presence of SYTOX, a membrane-impermeable DNA dye. HeLa cells deprived of amino acids for 15 h were fluorescence stained with histone H1 or IL6 antibodies ( D ), histone H2A, H2B, H3 or H4 antibodies ( E ) or HMGB1, Hsp90 or ERp57 antibodies ( F ) in combination with histone H1 antibody and 4',6-diamidino-2-phenylindole (DAPI). ( G ) Amino-acid-deprived HeLa cells were stained with SYTOX to determine viability, fixed and stained with DAPI and histone H1 antibodies. ( H ) Amino-acid-deprived HeLa cells were untreated or treated with MNase (500 mU/ml) for 10 min. Released DNA was quantitated at the indicated times. Data from triplicate samples are presented as mean±S.D. ( I ) Conditioned media from amino-acid-deprived HeLa cells treated or untreated with MNase were western blotted with histone H1, 2B, H3, H4, IL6, ERp57, HMGB1 or Hsp90 antibodies. ( J ) Images captured every hour from live imaging of amino-acid-deprived HeLa cells with SYTOX (green) and DRAQ5, membrane-permeable DNA dye (red). ( K ) SYTOX fluorescent intensities were measured from circularized areas of live imaging of amino-acid-deprived cells in 5-min intervals. ( L ) TEM images of control cells ( L a) and amino-acid-deprived HeLa cells ( L b– L d). ( M ) Amino-acid-deprived HeLa cells were fluorescence stained with lamin and nuclear pore antibodies, or lamin antibody and wheat germ agglutinin (WGE)

    Article Snippet: Released DNA from dying cells were digested with 500 mU/ml MNase (Thermo Scientific) for 5 min. Nuclease activity was stopped with 5 mM EDTA and the culture supernatants were collected and stored at −20 °C until quantification.

    Techniques: Genomic Sequencing, Polymerase Chain Reaction, Amplification, Fluorescence, Staining, Western Blot, Imaging, Transmission Electron Microscopy