Structured Review

New England Biolabs mnase
A possible model of low-level nucleoid organization. a A fragment of EM image of E. coli nucleoid (adapted from [ 21 ]). b Genomic <t>DNA</t> (gray) forms loops with A-tract clusters (cyan) located at apexes and <t>MNase</t> resistant fragments (black) occupying loop ‘stems’. c A blow-up of a DNA loop, containing several A-tracts (cyan) and two MNase resistant fragments (black)
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Images

1) Product Images from "Organization of DNA in a bacterial nucleoid"

Article Title: Organization of DNA in a bacterial nucleoid

Journal: BMC Microbiology

doi: 10.1186/s12866-016-0637-3

A possible model of low-level nucleoid organization. a A fragment of EM image of E. coli nucleoid (adapted from [ 21 ]). b Genomic DNA (gray) forms loops with A-tract clusters (cyan) located at apexes and MNase resistant fragments (black) occupying loop ‘stems’. c A blow-up of a DNA loop, containing several A-tracts (cyan) and two MNase resistant fragments (black)
Figure Legend Snippet: A possible model of low-level nucleoid organization. a A fragment of EM image of E. coli nucleoid (adapted from [ 21 ]). b Genomic DNA (gray) forms loops with A-tract clusters (cyan) located at apexes and MNase resistant fragments (black) occupying loop ‘stems’. c A blow-up of a DNA loop, containing several A-tracts (cyan) and two MNase resistant fragments (black)

Techniques Used:

Analysis of the DNA fragments from in vivo MNase digestion of nucleoid in wild type E. coli ( a ) and in vitro MNase digestion of purified genomic DNA ( b ) in agarose gel. a Wild type cells with the empty vector (lanes 2, 3) and MNase-expressing vector (lanes 4-8) were supplemented with arabinose (lane 6), CaCl 2 (lane 5) or both (lanes 3, 7, 8). Digestion reactions were stopped 1 minute (lane 7) or 5 minutes (lanes 3, 5, 8) after CaCl 2 was added. Lanes 1 and 9 show DNA molecular weight marker. b Lane 1, purified wild type genomic DNA; lanes 2-6, a time course of in vitro MNase digestion of the wild type genomic DNA; lane 7, DNA molecular marker
Figure Legend Snippet: Analysis of the DNA fragments from in vivo MNase digestion of nucleoid in wild type E. coli ( a ) and in vitro MNase digestion of purified genomic DNA ( b ) in agarose gel. a Wild type cells with the empty vector (lanes 2, 3) and MNase-expressing vector (lanes 4-8) were supplemented with arabinose (lane 6), CaCl 2 (lane 5) or both (lanes 3, 7, 8). Digestion reactions were stopped 1 minute (lane 7) or 5 minutes (lanes 3, 5, 8) after CaCl 2 was added. Lanes 1 and 9 show DNA molecular weight marker. b Lane 1, purified wild type genomic DNA; lanes 2-6, a time course of in vitro MNase digestion of the wild type genomic DNA; lane 7, DNA molecular marker

Techniques Used: In Vivo, In Vitro, Purification, Agarose Gel Electrophoresis, Plasmid Preparation, Expressing, Molecular Weight, Marker

Genome-wide distribution of sequenced tags. a Tag frequencies for the entire E. coli genome. Frequencies of tags mapped on the positive and negative strands are shown with red and blue bars respectively. a , c Schematic illustrations of tag cross-correlation ( b ) and auto-correlation analyses ( c ). MNase resistant fragments are shown with grey rectangles. Vertical red and blue arrows represent 5’-ends of the digestion fragments mapping to the DNA positive and negative strands respectively
Figure Legend Snippet: Genome-wide distribution of sequenced tags. a Tag frequencies for the entire E. coli genome. Frequencies of tags mapped on the positive and negative strands are shown with red and blue bars respectively. a , c Schematic illustrations of tag cross-correlation ( b ) and auto-correlation analyses ( c ). MNase resistant fragments are shown with grey rectangles. Vertical red and blue arrows represent 5’-ends of the digestion fragments mapping to the DNA positive and negative strands respectively

Techniques Used: Genome Wide

2) Product Images from "Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum"

Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum

Journal: EMBO Reports

doi: 10.15252/embr.201846331

Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.
Figure Legend Snippet: Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.

Techniques Used: Immunofluorescence, Staining, Isolation, Expressing, Purification, Agarose Gel Electrophoresis, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Transfection

3) Product Images from "HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism"

Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004652

Isolation of cross-linked ORF57-RNA complexes for HITS-CLIP analysis. Top Nitrocellulose membrane from an ORF57 immunoprecipitation performed under HITS-CLIP conditions. Cross-linked RNA fragments were end-labeled with 32 P to be visualized by Phosphoimager. Cells were induced to undergo lytic reactivation, exposed to UV and/or treated with high or low concentrations of MNase as indicated. The samples lacking an ORF57 antibody (lane 1) were precipitated with pre-bleed antibodies from the same rabbit. Lanes 6 and 7 are a dark exposure of lanes 4 and 5. The dashed white box indicates the position of the ORF57 complex cut from the membrane for library preparation. Bottom Western blot of the same HITS-CLIP samples shown in the top panel. Affinity purified rabbit anti-ORF57 was used to detect ORF57. Positions of molecular weight markers are shown on the left. On the right, a single asterisk marks the position of a contaminating ~37 kDa protein; double and triple asterisks mark positions of putative ORF57 homodimers and homotrimers bound to the same RNA. The doublet is likely due to an ORF57 cleavage product [ 104 ].
Figure Legend Snippet: Isolation of cross-linked ORF57-RNA complexes for HITS-CLIP analysis. Top Nitrocellulose membrane from an ORF57 immunoprecipitation performed under HITS-CLIP conditions. Cross-linked RNA fragments were end-labeled with 32 P to be visualized by Phosphoimager. Cells were induced to undergo lytic reactivation, exposed to UV and/or treated with high or low concentrations of MNase as indicated. The samples lacking an ORF57 antibody (lane 1) were precipitated with pre-bleed antibodies from the same rabbit. Lanes 6 and 7 are a dark exposure of lanes 4 and 5. The dashed white box indicates the position of the ORF57 complex cut from the membrane for library preparation. Bottom Western blot of the same HITS-CLIP samples shown in the top panel. Affinity purified rabbit anti-ORF57 was used to detect ORF57. Positions of molecular weight markers are shown on the left. On the right, a single asterisk marks the position of a contaminating ~37 kDa protein; double and triple asterisks mark positions of putative ORF57 homodimers and homotrimers bound to the same RNA. The doublet is likely due to an ORF57 cleavage product [ 104 ].

Techniques Used: Isolation, Cross-linking Immunoprecipitation, Immunoprecipitation, Labeling, Western Blot, Affinity Purification, Molecular Weight

4) Product Images from "RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF"

Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky562

Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.
Figure Legend Snippet: Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.

Techniques Used: Binding Assay, Genome Wide, Sequencing, Immunoprecipitation

5) Product Images from "CAL1 is the Drosophila CENP-A assembly factor"

Article Title: CAL1 is the Drosophila CENP-A assembly factor

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201305036

CAL1 is a CENP-A–specific nucleosome assembly factor. (A) CENP-A 101–225 –H4 was assembled on circular relaxed plasmid (pCR2.1-4 [CEN3 + CEN6]) using increasing amounts of CAL1 1–96 , CAL1 1–132 , or CAL1 1–160 in the presence of topoisomerase I. The extracted DNA samples were analyzed on agarose gel and stained with SYBR gold. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lane 3: CENP-A 101–225 –H4, H2A–H2B but no CAL1; lanes 4–7: relaxed plasmid and CENP-A 101–225 –H4, H2A–H2B with CAL1 1–96 ; lanes 8–11: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–132 ; lanes 12–15: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–160 . (B) CENP-A–containing nucleosomes and histone H3 nucleosomes were assembled using histone proteins, CAL1 1–160 , or yeast Nap1 and pGEM3Z-601 plasmid. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lanes 3, 6, and 9: mock reaction without CAL1 1–160 or Nap1; lanes 4 and 5: H3–H4, H2A–H2B with CAL1 1–160 ; lanes 7 and 8: CENP-A 101–225 –H4, H2A-H2B with CAL1 1–160 ; lanes 10 and 11: H3–H4, H2A–H2B with Nap1; lanes 12 and 13: CENP-A 101–225 –H4, H2A–H2B with Nap1. (C) CAL1-assembled CENP-A nucleosomes are negatively supercoiled. CENP-A nucleosomes (lane CENP-A) were assembled by CAL1 1–160 in the presence of topoisomerase I and H2A–H2B dimers, whereas histone H3 nucleosomes (lane H3) were assembled by Nap1. Controls are: supercoiled plasmid (superc.), relaxed plasmid (relaxed), and relaxed plasmid without histones or Nap1, but treated and processed as in the H3 and CENP-A nucleosome assembly reactions (mock). The samples were analyzed on agarose gels with or without 1 µg/ml chloroquine. Boxes highlight the decrease in migration that occurs in the presence of chloroquine in both H3 and CENP-A nucleosomes. (A–C) S, supercoiled; R, relaxed plasmid. (D) Diagram depicting the expected migration patterns for negatively and positively supercoiled DNA separated by 2D gel electrophoresis ( Tachiwana et al., 2011 ). Left gel: H3 nucleosomes assembled with Nap1; right gel: CENP-A nucleosomes assembled with CAL1 1–160 . (E) Mononucleosomes were assembled on 147-bp Widom DNA with Nap1 or CAL1 1–160 and digested with MNase for 30 or 120 s. Native PAGE of samples shown. Lanes 1–3: DNA only; lanes 4 and 5: H3 nucleosomes assembled by Nap1; lanes 6 and 7: CENP-A nucleosomes assembled by Nap1; lanes 8 and 9: CENP-A nucleosomes assembled by CAL1 1–160 .
Figure Legend Snippet: CAL1 is a CENP-A–specific nucleosome assembly factor. (A) CENP-A 101–225 –H4 was assembled on circular relaxed plasmid (pCR2.1-4 [CEN3 + CEN6]) using increasing amounts of CAL1 1–96 , CAL1 1–132 , or CAL1 1–160 in the presence of topoisomerase I. The extracted DNA samples were analyzed on agarose gel and stained with SYBR gold. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lane 3: CENP-A 101–225 –H4, H2A–H2B but no CAL1; lanes 4–7: relaxed plasmid and CENP-A 101–225 –H4, H2A–H2B with CAL1 1–96 ; lanes 8–11: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–132 ; lanes 12–15: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–160 . (B) CENP-A–containing nucleosomes and histone H3 nucleosomes were assembled using histone proteins, CAL1 1–160 , or yeast Nap1 and pGEM3Z-601 plasmid. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lanes 3, 6, and 9: mock reaction without CAL1 1–160 or Nap1; lanes 4 and 5: H3–H4, H2A–H2B with CAL1 1–160 ; lanes 7 and 8: CENP-A 101–225 –H4, H2A-H2B with CAL1 1–160 ; lanes 10 and 11: H3–H4, H2A–H2B with Nap1; lanes 12 and 13: CENP-A 101–225 –H4, H2A–H2B with Nap1. (C) CAL1-assembled CENP-A nucleosomes are negatively supercoiled. CENP-A nucleosomes (lane CENP-A) were assembled by CAL1 1–160 in the presence of topoisomerase I and H2A–H2B dimers, whereas histone H3 nucleosomes (lane H3) were assembled by Nap1. Controls are: supercoiled plasmid (superc.), relaxed plasmid (relaxed), and relaxed plasmid without histones or Nap1, but treated and processed as in the H3 and CENP-A nucleosome assembly reactions (mock). The samples were analyzed on agarose gels with or without 1 µg/ml chloroquine. Boxes highlight the decrease in migration that occurs in the presence of chloroquine in both H3 and CENP-A nucleosomes. (A–C) S, supercoiled; R, relaxed plasmid. (D) Diagram depicting the expected migration patterns for negatively and positively supercoiled DNA separated by 2D gel electrophoresis ( Tachiwana et al., 2011 ). Left gel: H3 nucleosomes assembled with Nap1; right gel: CENP-A nucleosomes assembled with CAL1 1–160 . (E) Mononucleosomes were assembled on 147-bp Widom DNA with Nap1 or CAL1 1–160 and digested with MNase for 30 or 120 s. Native PAGE of samples shown. Lanes 1–3: DNA only; lanes 4 and 5: H3 nucleosomes assembled by Nap1; lanes 6 and 7: CENP-A nucleosomes assembled by Nap1; lanes 8 and 9: CENP-A nucleosomes assembled by CAL1 1–160 .

Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Staining, Migration, Two-Dimensional Gel Electrophoresis, Electrophoresis, Clear Native PAGE

6) Product Images from "RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF"

Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky562

Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.
Figure Legend Snippet: Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.

Techniques Used: Binding Assay, Genome Wide, Sequencing, Immunoprecipitation

7) Product Images from "Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics"

Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky526

Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.
Figure Legend Snippet: Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.

Techniques Used: Clear Native PAGE, Electrophoresis, Polyacrylamide Gel Electrophoresis, Staining

8) Product Images from "Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics"

Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky526

Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.
Figure Legend Snippet: Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.

Techniques Used: Clear Native PAGE, Electrophoresis, Polyacrylamide Gel Electrophoresis, Staining

9) Product Images from "Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure"

Article Title: Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku150

Nucleosome duplexes are decoupled into mononucleosomes. ( A ) EMSA of purified nucleosome duplexes containing H3(T118ph) HO and mp2-187 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S4 ). ( B ) Quantification of fraction of nucleosome duplexes (squares), nucleosome (circles) and free DNA (diamond) species for the gel in (A) versus time. Error bars are the standard deviation of three independent experiments. ( C ) EMSA of purified nucleosome duplexes and altosomes containing H3(T118ph) HO and mp2-247 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert in part to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S5 ). ( D ) Quantification of the fraction of nucleosome duplexes (squares), altosomes (triangles), nucleosome (circles) and free DNA (diamond) species for the gel in (C) versus time. Error bars are the standard deviation of three independent experiments.
Figure Legend Snippet: Nucleosome duplexes are decoupled into mononucleosomes. ( A ) EMSA of purified nucleosome duplexes containing H3(T118ph) HO and mp2-187 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S4 ). ( B ) Quantification of fraction of nucleosome duplexes (squares), nucleosome (circles) and free DNA (diamond) species for the gel in (A) versus time. Error bars are the standard deviation of three independent experiments. ( C ) EMSA of purified nucleosome duplexes and altosomes containing H3(T118ph) HO and mp2-247 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert in part to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S5 ). ( D ) Quantification of the fraction of nucleosome duplexes (squares), altosomes (triangles), nucleosome (circles) and free DNA (diamond) species for the gel in (C) versus time. Error bars are the standard deviation of three independent experiments.

Techniques Used: Purification, Standard Deviation

10) Product Images from "Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages"

Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages

Journal: PLoS ONE

doi: 10.1371/journal.pone.0093971

PolII and TBP binding in the fraction of IL12B and IL1A promoters in a population of induced BMDMs that is nucleosome-free. (A and B), ChIP experiments were performed as described in the legend of Figure 2F with antibodies that detect (A) PolII or (B) TBP in BMDMs before (dark blue bars), and upon 1.5 h (yellow) or 3 h (red) LPS induction. Cross-linked chromatin was either untreated (solid bars), or lightly digested with MNase (hatched bars) as described in the Materials and Methods . The data was normalized to a region in the KIT promoter and genomic locations are indicated. The experiment was performed twice and error bars indicating the SEM are shown.
Figure Legend Snippet: PolII and TBP binding in the fraction of IL12B and IL1A promoters in a population of induced BMDMs that is nucleosome-free. (A and B), ChIP experiments were performed as described in the legend of Figure 2F with antibodies that detect (A) PolII or (B) TBP in BMDMs before (dark blue bars), and upon 1.5 h (yellow) or 3 h (red) LPS induction. Cross-linked chromatin was either untreated (solid bars), or lightly digested with MNase (hatched bars) as described in the Materials and Methods . The data was normalized to a region in the KIT promoter and genomic locations are indicated. The experiment was performed twice and error bars indicating the SEM are shown.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation

11) Product Images from "An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps"

Article Title: An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2013.00166

DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.
Figure Legend Snippet: DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.

Techniques Used: Migration, Incubation, Agarose Gel Electrophoresis

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Filtration:

Article Title: Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells
Article Snippet: Chromatin pellets were briefly digested with MNase (New England BioLabs) and the mononucleosomes were monitored by electrophoresis 0.5ug anti-H2A.X antibody (active motif 39689) was used per ChIP experiment. .. After filtration, the qualified tags (in fastq format) were aligned to the mouse genome (mm9) with bowtie2 (2.0.6) .

Electrophoresis:

Article Title: Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells
Article Snippet: .. Chromatin pellets were briefly digested with MNase (New England BioLabs) and the mononucleosomes were monitored by electrophoresis 0.5ug anti-H2A.X antibody (active motif 39689) was used per ChIP experiment. .. Co-purified DNA and whole cell extraction (WCE) input genomic DNA were subject to library construction, cluster generation and next-generation sequencing (Illumina Genome Analyzer II and HiSeq 2000).

Article Title: An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps
Article Snippet: NET digestion by nucleases In a first set of experiments, 4 μg of purified λDNA (Invitrogen) was treated with 4 U/mL DNase I (Sigma Aldrich), 4 U/mL MNase (New England BioLabs, France), or 4 U/mL Alu I (New England BioLabs) for 20 min at 37°C. .. Electrophoresis was run at 100 V for 30 min and DNA was visualized with an ultraviolet transilluminator (MiniBIS-Pro, DNR Bio-imaging Systems).

Incubation:

Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
Article Snippet: To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA. .. Digested chromatin was diluted 3-fold with High Salt ChIP buffer (10 mM Tris-HCl, pH 8, 400 mM NaCl, 1% TritonX-100, 2 mM EDTA, Complete protease inhibitors w/o EDTA (Roche)) to yield 600 μl total volume and incubated overnight at 4°C with either 5 μl of anti-H3 (39163, Active Motif, concentration is not known), 4 μg of anti-H2A.Z (ab4174; Abcam), 1 μg of anti-H3K4me1 (ab8895; Abcam), 1 μg of anti-H3K4me3 (ab8580; Abcam) or 1 μg of H3K27ac (ab4729; Abcam).

Article Title: CAL1 is the Drosophila CENP-A assembly factor
Article Snippet: To assemble CENP-A nucleosomes, Drosophila CENP-A101–225 –H4–Cal11–160 (3.8 µM), Alexa Fluor 488–labeled H2A–H2B (2.5 µM), and 147-bp Widom 601 DNA (0.72 µM) were mixed and incubated for 1 h under low salt buffer conditions. .. MNase digestion of nucleosomes was conducted by incubating nucleosomes (∼60 nM) with 2,000 U/ml MNase (New England Biolabs, Inc.) for 30–120 s. After the reactions were stopped by addition of 20 mM EDTA, the samples were treated with 2% SDS and 0.4 mg/ml proteinase K for 30 min at 55°C.

Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism
Article Snippet: Next, CaCl2 was added to 5 mM with 30 U of RQ1 DNase (Promega) and incubated for 15 min at 25°C. .. For RNA digestion, MNase (New England Biolabs) was diluted to 10 gel units/μL in RIPA buffer and 5 μL of this freshly diluted 1:200 stock was added to the extract.

Article Title: Identification of the PTEN-ARID4B-PI3K pathway reveals the dependency on ARID4B by PTEN-deficient prostate cancer
Article Snippet: .. From the resuspended solutions, 100 µl aliquots were taken and incubated with or without MNase (100 units) (New England Biolabs) for 5 min. .. The reaction was stopped by adding 80 µl of MNase digestion buffer, 20 µl of MNase stop buffer (100 mM EDTA, 10 mM EGTA), 3 µl of proteinase K (25 mg/ml) (Roche), and 10 µl of 20% SDS, and then incubated overnight at 37 °C.

Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum
Article Snippet: Briefly, schizont‐stage parasites of WT or WT + PfH3p‐HA were isolated from infected erythrocytes by saponin lysis, resuspended in 1 ml of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 ) supplemented with protease inhibitors (Complete, Roche), and incubated for 30 min at 4°C. .. Mononucleosomes were prepared from freshly isolated nuclei by digestion with 4 U MNase (NEB) for different amounts of time at 33°C; digestion was stopped by adding 10 mM EGTA pH 8.0.

Article Title: Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest
Article Snippet: Pellets were washed once with nuclease buffer (50 mM Tris pH 8.0, 5 mM CaCl2 , and 100 µg/ml BSA), split in two, and resuspended in nuclease buffer alone or nuclease buffer containing 200 U/ml microccocal nuclease (New England Biolabs, Ipswich, MA, USA). .. Pellets were incubated for 30 minutes on ice and then centrifuged at 12000g for 10 minutes.

Gel Extraction:

Article Title: Organization of DNA in a bacterial nucleoid
Article Snippet: RNAs and proteins were removed from the sample by intensive treatment first with RNase A and then with proteinase K. Resultant samples were run in 1.2 % agarose gel and the band containing the shortest DNA digestion fragments (around 100 bp) was cut out for subsequent DNA purification with QIAquick Gel Extraction Kit (Qiagen) and sequencing. .. The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

Western Blot:

Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF
Article Snippet: Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp. .. Flag-RBPJ was precipitated using Flag-M2 agarose beads (Sigma) and washed immunoprecipates were subjected to western blotting using Abs to Flag or HistoneH3 (sc-8654) from Santa Cruz.

Transfection:

Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF
Article Snippet: To quantify RBPJ binding to nucleosomes, Flag-RBPJ was transfected into 293 cells. .. Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

Fluorescence:

Article Title: CAL1 is the Drosophila CENP-A assembly factor
Article Snippet: The nucleosomes were analyzed by 6% native PAGE and stained by SYBR gold; the incorporation of H2A–H2B was confirmed by fluorescence imaging of the gel for Alexa Fluor 488. .. MNase digestion of nucleosomes was conducted by incubating nucleosomes (∼60 nM) with 2,000 U/ml MNase (New England Biolabs, Inc.) for 30–120 s. After the reactions were stopped by addition of 20 mM EDTA, the samples were treated with 2% SDS and 0.4 mg/ml proteinase K for 30 min at 55°C.

Concentration Assay:

Article Title: Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure
Article Snippet: Reactions were carried out in an initial volume of 10 μl with 10 nM nucleosomes and 0–40 U/ml of MNase (New England Biolabs) in 20 mM Tris, pH 8.0, 0.5 mM CaCl2 at 37°C to prevent H3(T118ph) nucleosome disassembly. .. After 20 min, reactions were quenched with 15 mM EDTA final concentration and resolved by PAGE.

Article Title: Organization of DNA in a bacterial nucleoid
Article Snippet: To quench the reactions 12 μl of 10 % solution of phenol in ethanol and EGTA to final concentration 25 mM were added on ice to each aliquot. .. The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
Article Snippet: To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA. .. Digested chromatin was diluted 3-fold with High Salt ChIP buffer (10 mM Tris-HCl, pH 8, 400 mM NaCl, 1% TritonX-100, 2 mM EDTA, Complete protease inhibitors w/o EDTA (Roche)) to yield 600 μl total volume and incubated overnight at 4°C with either 5 μl of anti-H3 (39163, Active Motif, concentration is not known), 4 μg of anti-H2A.Z (ab4174; Abcam), 1 μg of anti-H3K4me1 (ab8895; Abcam), 1 μg of anti-H3K4me3 (ab8580; Abcam) or 1 μg of H3K27ac (ab4729; Abcam).

Protease Inhibitor:

Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism
Article Snippet: The composition of the SDS lysis buffer was 0.5% SDS, 50mM Tris pH 6.8, 1mM EDTA, 0.125 mg/ml Heparin, 1mM DTT, 1mM PMSF and 1X protease inhibitor (Calbiochem), while RIPA correction buffer was 1.25% NP40, 0.625% sodium deoxycholate, 62.5 mM Tris pH 8.0, 2.25 mM EDTA, 187.5 mM NaCl, 0.125 mg/ml Heparin, 1mM DTT, 1mM PMSF, with 1X protease inhibitors. .. For RNA digestion, MNase (New England Biolabs) was diluted to 10 gel units/μL in RIPA buffer and 5 μL of this freshly diluted 1:200 stock was added to the extract.

Footprinting:

Article Title: Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure
Article Snippet: Paragraph title: MNase footprinting ... Reactions were carried out in an initial volume of 10 μl with 10 nM nucleosomes and 0–40 U/ml of MNase (New England Biolabs) in 20 mM Tris, pH 8.0, 0.5 mM CaCl2 at 37°C to prevent H3(T118ph) nucleosome disassembly.

Infection:

Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum
Article Snippet: Briefly, schizont‐stage parasites of WT or WT + PfH3p‐HA were isolated from infected erythrocytes by saponin lysis, resuspended in 1 ml of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 ) supplemented with protease inhibitors (Complete, Roche), and incubated for 30 min at 4°C. .. Mononucleosomes were prepared from freshly isolated nuclei by digestion with 4 U MNase (NEB) for different amounts of time at 33°C; digestion was stopped by adding 10 mM EGTA pH 8.0.

Imaging:

Article Title: CAL1 is the Drosophila CENP-A assembly factor
Article Snippet: The nucleosomes were analyzed by 6% native PAGE and stained by SYBR gold; the incorporation of H2A–H2B was confirmed by fluorescence imaging of the gel for Alexa Fluor 488. .. MNase digestion of nucleosomes was conducted by incubating nucleosomes (∼60 nM) with 2,000 U/ml MNase (New England Biolabs, Inc.) for 30–120 s. After the reactions were stopped by addition of 20 mM EDTA, the samples were treated with 2% SDS and 0.4 mg/ml proteinase K for 30 min at 55°C.

Polymerase Chain Reaction:

Article Title: Organization of DNA in a bacterial nucleoid
Article Snippet: The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C. .. DNA fragments with sizes comparable to those from in vivo digestion were purified directly from the reaction with QIAquick PCR Purification Kit (Qiagen).

Sonication:

Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF
Article Snippet: For ChIP-PCR and for RNA Pol II and H3K9ac ChIP-seq, the MNAse digestion step was replaced with a sonication step in 0.12% SDS followed by 5× dilution with concentrated ChIP buffer. .. Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
Article Snippet: Chromatin immunoprecipitation Chromatin from 5×106 cells per antibody that had been cross-linked with 0.5% formaldehyde for 10 min was isolated by sonication with a Branson sonifier (10 pulses of 10″ at setting 4) in Lysis buffer (50 mM Hepes-KOH, pH 7.5, 1% TritonX-100, 0.1% SDS) and centrifugation for 10′ at 21,000×g . .. To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA.

Binding Assay:

Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF
Article Snippet: To quantify RBPJ binding to nucleosomes, Flag-RBPJ was transfected into 293 cells. .. Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
Article Snippet: .. To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA. .. Digested chromatin was diluted 3-fold with High Salt ChIP buffer (10 mM Tris-HCl, pH 8, 400 mM NaCl, 1% TritonX-100, 2 mM EDTA, Complete protease inhibitors w/o EDTA (Roche)) to yield 600 μl total volume and incubated overnight at 4°C with either 5 μl of anti-H3 (39163, Active Motif, concentration is not known), 4 μg of anti-H2A.Z (ab4174; Abcam), 1 μg of anti-H3K4me1 (ab8895; Abcam), 1 μg of anti-H3K4me3 (ab8580; Abcam) or 1 μg of H3K27ac (ab4729; Abcam).

Article Title: Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest
Article Snippet: Paragraph title: Chromatin binding assays ... Pellets were washed once with nuclease buffer (50 mM Tris pH 8.0, 5 mM CaCl2 , and 100 µg/ml BSA), split in two, and resuspended in nuclease buffer alone or nuclease buffer containing 200 U/ml microccocal nuclease (New England Biolabs, Ipswich, MA, USA).

ChIP-sequencing:

Article Title: Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells
Article Snippet: Briefly, approximately 10 million ESCs were used for each ChIP and massive parallel sequencing (ChIP-Seq) experiment. .. Chromatin pellets were briefly digested with MNase (New England BioLabs) and the mononucleosomes were monitored by electrophoresis 0.5ug anti-H2A.X antibody (active motif 39689) was used per ChIP experiment.

Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF
Article Snippet: Paragraph title: ChIP-seq and ChIP-PCR ... Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

In Vivo:

Article Title: Organization of DNA in a bacterial nucleoid
Article Snippet: Paragraph title: In vivo MNase digestion ... The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

Sensitive Assay:

Article Title: Identification of the PTEN-ARID4B-PI3K pathway reveals the dependency on ARID4B by PTEN-deficient prostate cancer
Article Snippet: Paragraph title: Micrococcal nuclease (MNase) sensitivity assay ... From the resuspended solutions, 100 µl aliquots were taken and incubated with or without MNase (100 units) (New England Biolabs) for 5 min.

Magnetic Beads:

Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
Article Snippet: To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA. .. 20 μl of Protein A/G magnetic beads (Pierce) were added to the reaction and incubated at 4°C for 2 h. Beads were washed with 280 μl each of TSE buffer (20 mM Tris pH 8.0, 0.1% SDS, 1% TritonX-100, 2 mM EDTA), TSE250 (TSE buffer, 250 mM NaCl) and TSE500 (TSE buffer, 500 mM NaCl), Wash buffer III (10 mM Tris pH 8.5, 0.25 M LiCl, 1% NP-40/Igepal, 1% deoxycholate, 1 mM EDTA) and TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) all containing Complete protease inhibitors.

Isolation:

Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
Article Snippet: .. To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA. .. Digested chromatin was diluted 3-fold with High Salt ChIP buffer (10 mM Tris-HCl, pH 8, 400 mM NaCl, 1% TritonX-100, 2 mM EDTA, Complete protease inhibitors w/o EDTA (Roche)) to yield 600 μl total volume and incubated overnight at 4°C with either 5 μl of anti-H3 (39163, Active Motif, concentration is not known), 4 μg of anti-H2A.Z (ab4174; Abcam), 1 μg of anti-H3K4me1 (ab8895; Abcam), 1 μg of anti-H3K4me3 (ab8580; Abcam) or 1 μg of H3K27ac (ab4729; Abcam).

Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum
Article Snippet: .. Mononucleosomes were prepared from freshly isolated nuclei by digestion with 4 U MNase (NEB) for different amounts of time at 33°C; digestion was stopped by adding 10 mM EGTA pH 8.0. .. After spinning out debris at 18,800 g for 15 min at 4°C, the supernatant was analyzed for the presence of mononucleosomes by extracting DNA and resolving it on a 2% agarose gel stained with ethidium bromide.

Clarification Assay:

Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism
Article Snippet: For RNA digestion, MNase (New England Biolabs) was diluted to 10 gel units/μL in RIPA buffer and 5 μL of this freshly diluted 1:200 stock was added to the extract. .. After clarification of the lysate by three successive centrifugation steps at 21K x g for 10 min, the lysate was pre-cleared and the ORF57-RNA complexes were immunoprecipitated with protein A Dynabeads (Invitrogen).

Purification:

Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics
Article Snippet: Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C. .. DNA was purified and resolved on 6.5% polyacrylamide gel (37.5:1) in 1× TAE buffer (40 mM Tris-OH, 20.6 mM acetic acid, 5 mM sodium acetate, 2 mM EDTA) and stained with SYBR Gold.

Article Title: Organization of DNA in a bacterial nucleoid
Article Snippet: .. The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C. ..

Article Title: An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps
Article Snippet: .. NET digestion by nucleases In a first set of experiments, 4 μg of purified λDNA (Invitrogen) was treated with 4 U/mL DNase I (Sigma Aldrich), 4 U/mL MNase (New England BioLabs, France), or 4 U/mL Alu I (New England BioLabs) for 20 min at 37°C. .. The samples were then loaded on 0.8% agarose gels (w/v) prepared in Tris-acetate-EDTA buffer containing 0.5 μg/mL ethidium bromide (Invitrogen).

Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics
Article Snippet: Micrococcal nuclease digestion Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C. .. DNA was purified and resolved on 6.5% polyacrylamide gel (37.5:1) in 1× TAE buffer (40 mM Tris-OH, 20.6 mM acetic acid, 5 mM sodium acetate, 2 mM EDTA) and stained with SYBR Gold.

Article Title: Identification of the PTEN-ARID4B-PI3K pathway reveals the dependency on ARID4B by PTEN-deficient prostate cancer
Article Snippet: From the resuspended solutions, 100 µl aliquots were taken and incubated with or without MNase (100 units) (New England Biolabs) for 5 min. .. The purified DNA was subjected to qPCR analysis using the primer sets described for ChIP-qPCR analysis.

Sequencing:

Article Title: Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells
Article Snippet: Briefly, approximately 10 million ESCs were used for each ChIP and massive parallel sequencing (ChIP-Seq) experiment. .. Chromatin pellets were briefly digested with MNase (New England BioLabs) and the mononucleosomes were monitored by electrophoresis 0.5ug anti-H2A.X antibody (active motif 39689) was used per ChIP experiment.

Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF
Article Snippet: DNA samples for high throughput sequencing were prepared by the UNC genomics core and 50bp or 100 bp paired ends sequencing was performed using the Illumina platform. .. Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

Article Title: Organization of DNA in a bacterial nucleoid
Article Snippet: As control in sequence analysis, we used DNA fragments obtained from in vitro MNase digestion of purified wild type genomic DNA. .. The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

Article Title: CAL1 is the Drosophila CENP-A assembly factor
Article Snippet: MNase protection assays CENP-A nucleosomes were assembled by Nap1 or CAL11–160 under low salt buffer conditions (10 mM Tris-HCl, pH 7.5, 270 mM NaCl, 2% glycerol, 60 µg/ml BSA, 0.75 mM EDTA, and 0.1 mM DTT) on the 147-bp Widom 601 DNA sequence. .. MNase digestion of nucleosomes was conducted by incubating nucleosomes (∼60 nM) with 2,000 U/ml MNase (New England Biolabs, Inc.) for 30–120 s. After the reactions were stopped by addition of 20 mM EDTA, the samples were treated with 2% SDS and 0.4 mg/ml proteinase K for 30 min at 55°C.

Polyacrylamide Gel Electrophoresis:

Article Title: Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure
Article Snippet: Reactions were carried out in an initial volume of 10 μl with 10 nM nucleosomes and 0–40 U/ml of MNase (New England Biolabs) in 20 mM Tris, pH 8.0, 0.5 mM CaCl2 at 37°C to prevent H3(T118ph) nucleosome disassembly. .. After 20 min, reactions were quenched with 15 mM EDTA final concentration and resolved by PAGE.

Nuclease Assay:

Article Title: Identification of the PTEN-ARID4B-PI3K pathway reveals the dependency on ARID4B by PTEN-deficient prostate cancer
Article Snippet: Micrococcal nuclease (MNase) sensitivity assay Micrococcal nuclease assay was performed as described . .. From the resuspended solutions, 100 µl aliquots were taken and incubated with or without MNase (100 units) (New England Biolabs) for 5 min.

Chromatin Immunoprecipitation:

Article Title: Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells
Article Snippet: .. Chromatin pellets were briefly digested with MNase (New England BioLabs) and the mononucleosomes were monitored by electrophoresis 0.5ug anti-H2A.X antibody (active motif 39689) was used per ChIP experiment. .. Co-purified DNA and whole cell extraction (WCE) input genomic DNA were subject to library construction, cluster generation and next-generation sequencing (Illumina Genome Analyzer II and HiSeq 2000).

Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF
Article Snippet: Paragraph title: ChIP-seq and ChIP-PCR ... Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

Article Title: Targeting the histone methyltransferase G9a activates imprinted genes and improves survival of a mouse model of Prader–Willi syndrome
Article Snippet: After rinsing the cell pellets, we added MNase (NEB) to digest the open status of chromatins, followed by genomic qPCR to determine changes in amount of SNRPN and other imprinted genes. .. The primers that we used in this study are described under ‘Chromatin immunoprecipitation assay.’

Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
Article Snippet: .. To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA. .. Digested chromatin was diluted 3-fold with High Salt ChIP buffer (10 mM Tris-HCl, pH 8, 400 mM NaCl, 1% TritonX-100, 2 mM EDTA, Complete protease inhibitors w/o EDTA (Roche)) to yield 600 μl total volume and incubated overnight at 4°C with either 5 μl of anti-H3 (39163, Active Motif, concentration is not known), 4 μg of anti-H2A.Z (ab4174; Abcam), 1 μg of anti-H3K4me1 (ab8895; Abcam), 1 μg of anti-H3K4me3 (ab8580; Abcam) or 1 μg of H3K27ac (ab4729; Abcam).

Article Title: Identification of the PTEN-ARID4B-PI3K pathway reveals the dependency on ARID4B by PTEN-deficient prostate cancer
Article Snippet: From the resuspended solutions, 100 µl aliquots were taken and incubated with or without MNase (100 units) (New England Biolabs) for 5 min. .. The purified DNA was subjected to qPCR analysis using the primer sets described for ChIP-qPCR analysis.

Real-time Polymerase Chain Reaction:

Article Title: Targeting the histone methyltransferase G9a activates imprinted genes and improves survival of a mouse model of Prader–Willi syndrome
Article Snippet: .. After rinsing the cell pellets, we added MNase (NEB) to digest the open status of chromatins, followed by genomic qPCR to determine changes in amount of SNRPN and other imprinted genes. ..

Article Title: Identification of the PTEN-ARID4B-PI3K pathway reveals the dependency on ARID4B by PTEN-deficient prostate cancer
Article Snippet: From the resuspended solutions, 100 µl aliquots were taken and incubated with or without MNase (100 units) (New England Biolabs) for 5 min. .. The purified DNA was subjected to qPCR analysis using the primer sets described for ChIP-qPCR analysis.

Agarose Gel Electrophoresis:

Article Title: Organization of DNA in a bacterial nucleoid
Article Snippet: RNAs and proteins were removed from the sample by intensive treatment first with RNase A and then with proteinase K. Resultant samples were run in 1.2 % agarose gel and the band containing the shortest DNA digestion fragments (around 100 bp) was cut out for subsequent DNA purification with QIAquick Gel Extraction Kit (Qiagen) and sequencing. .. The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum
Article Snippet: Mononucleosomes were prepared from freshly isolated nuclei by digestion with 4 U MNase (NEB) for different amounts of time at 33°C; digestion was stopped by adding 10 mM EGTA pH 8.0. .. After spinning out debris at 18,800 g for 15 min at 4°C, the supernatant was analyzed for the presence of mononucleosomes by extracting DNA and resolving it on a 2% agarose gel stained with ethidium bromide.

In Vitro:

Article Title: Organization of DNA in a bacterial nucleoid
Article Snippet: As control in sequence analysis, we used DNA fragments obtained from in vitro MNase digestion of purified wild type genomic DNA. .. The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

Ethanol Precipitation:

Article Title: Identification of the PTEN-ARID4B-PI3K pathway reveals the dependency on ARID4B by PTEN-deficient prostate cancer
Article Snippet: From the resuspended solutions, 100 µl aliquots were taken and incubated with or without MNase (100 units) (New England Biolabs) for 5 min. .. This was followed by phenol–chloroform extraction and ethanol precipitation.

Next-Generation Sequencing:

Article Title: Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells
Article Snippet: Chromatin pellets were briefly digested with MNase (New England BioLabs) and the mononucleosomes were monitored by electrophoresis 0.5ug anti-H2A.X antibody (active motif 39689) was used per ChIP experiment. .. Co-purified DNA and whole cell extraction (WCE) input genomic DNA were subject to library construction, cluster generation and next-generation sequencing (Illumina Genome Analyzer II and HiSeq 2000).

Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF
Article Snippet: DNA samples for high throughput sequencing were prepared by the UNC genomics core and 50bp or 100 bp paired ends sequencing was performed using the Illumina platform. .. Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

Immunoprecipitation:

Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism
Article Snippet: Paragraph title: HITS-CLIP lysate preparation and immunoprecipitation ... For RNA digestion, MNase (New England Biolabs) was diluted to 10 gel units/μL in RIPA buffer and 5 μL of this freshly diluted 1:200 stock was added to the extract.

N-ChIP:

Article Title: Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells
Article Snippet: Paragraph title: Native Chromatin immunoprecipitation (N-ChIP) ... Chromatin pellets were briefly digested with MNase (New England BioLabs) and the mononucleosomes were monitored by electrophoresis 0.5ug anti-H2A.X antibody (active motif 39689) was used per ChIP experiment.

DNA Purification:

Article Title: Organization of DNA in a bacterial nucleoid
Article Snippet: .. The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C. ..

Lysis:

Article Title: Targeting the histone methyltransferase G9a activates imprinted genes and improves survival of a mouse model of Prader–Willi syndrome
Article Snippet: Briefly, 3 d after drug treatment in human PWS fibroblasts, we harvested the cells and lysed with lysis buffer (0.5% NP-40, 15 mM Tris-HCl (pH 7.4), 0.15 mM spermidine, 0.5 mM spermine, 15 mM NaCl, 60 mM KCl, 1 mM DTT, 0.1mM PMSF, 0.5-M sucrose, protease and phosphatase inhibitor cocktail (Roche)). .. After rinsing the cell pellets, we added MNase (NEB) to digest the open status of chromatins, followed by genomic qPCR to determine changes in amount of SNRPN and other imprinted genes.

Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
Article Snippet: Chromatin immunoprecipitation Chromatin from 5×106 cells per antibody that had been cross-linked with 0.5% formaldehyde for 10 min was isolated by sonication with a Branson sonifier (10 pulses of 10″ at setting 4) in Lysis buffer (50 mM Hepes-KOH, pH 7.5, 1% TritonX-100, 0.1% SDS) and centrifugation for 10′ at 21,000×g . .. To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA.

Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism
Article Snippet: The composition of the SDS lysis buffer was 0.5% SDS, 50mM Tris pH 6.8, 1mM EDTA, 0.125 mg/ml Heparin, 1mM DTT, 1mM PMSF and 1X protease inhibitor (Calbiochem), while RIPA correction buffer was 1.25% NP40, 0.625% sodium deoxycholate, 62.5 mM Tris pH 8.0, 2.25 mM EDTA, 187.5 mM NaCl, 0.125 mg/ml Heparin, 1mM DTT, 1mM PMSF, with 1X protease inhibitors. .. For RNA digestion, MNase (New England Biolabs) was diluted to 10 gel units/μL in RIPA buffer and 5 μL of this freshly diluted 1:200 stock was added to the extract.

Article Title: Identification of the PTEN-ARID4B-PI3K pathway reveals the dependency on ARID4B by PTEN-deficient prostate cancer
Article Snippet: Briefly, control and ARID4B KO PC3 cells were resuspended in ice-cold lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2 , 0.5% Nonidet P-40, 0.15 mM spermine and 0.5 mM spermidine], and the nuclei were repelleted. .. From the resuspended solutions, 100 µl aliquots were taken and incubated with or without MNase (100 units) (New England Biolabs) for 5 min.

Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum
Article Snippet: Briefly, schizont‐stage parasites of WT or WT + PfH3p‐HA were isolated from infected erythrocytes by saponin lysis, resuspended in 1 ml of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 ) supplemented with protease inhibitors (Complete, Roche), and incubated for 30 min at 4°C. .. Mononucleosomes were prepared from freshly isolated nuclei by digestion with 4 U MNase (NEB) for different amounts of time at 33°C; digestion was stopped by adding 10 mM EGTA pH 8.0.

Article Title: Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest
Article Snippet: Chromatin binding assays Cells were lysed in triton lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% Triton X-100, and complete protease inhibitors cocktail (Roche) for 15 minutes. .. Pellets were washed once with nuclease buffer (50 mM Tris pH 8.0, 5 mM CaCl2 , and 100 µg/ml BSA), split in two, and resuspended in nuclease buffer alone or nuclease buffer containing 200 U/ml microccocal nuclease (New England Biolabs, Ipswich, MA, USA).

Staining:

Article Title: Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure
Article Snippet: Reactions were carried out in an initial volume of 10 μl with 10 nM nucleosomes and 0–40 U/ml of MNase (New England Biolabs) in 20 mM Tris, pH 8.0, 0.5 mM CaCl2 at 37°C to prevent H3(T118ph) nucleosome disassembly. .. Gels were stained with SYBR Gold (Invitrogen) and imaged by a Typhoon 8600 variable mode imager (GE Healthcare).

Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics
Article Snippet: Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C. .. DNA was purified and resolved on 6.5% polyacrylamide gel (37.5:1) in 1× TAE buffer (40 mM Tris-OH, 20.6 mM acetic acid, 5 mM sodium acetate, 2 mM EDTA) and stained with SYBR Gold.

Article Title: CAL1 is the Drosophila CENP-A assembly factor
Article Snippet: The nucleosomes were analyzed by 6% native PAGE and stained by SYBR gold; the incorporation of H2A–H2B was confirmed by fluorescence imaging of the gel for Alexa Fluor 488. .. MNase digestion of nucleosomes was conducted by incubating nucleosomes (∼60 nM) with 2,000 U/ml MNase (New England Biolabs, Inc.) for 30–120 s. After the reactions were stopped by addition of 20 mM EDTA, the samples were treated with 2% SDS and 0.4 mg/ml proteinase K for 30 min at 55°C.

Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics
Article Snippet: Micrococcal nuclease digestion Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C. .. DNA was purified and resolved on 6.5% polyacrylamide gel (37.5:1) in 1× TAE buffer (40 mM Tris-OH, 20.6 mM acetic acid, 5 mM sodium acetate, 2 mM EDTA) and stained with SYBR Gold.

Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum
Article Snippet: Mononucleosomes were prepared from freshly isolated nuclei by digestion with 4 U MNase (NEB) for different amounts of time at 33°C; digestion was stopped by adding 10 mM EGTA pH 8.0. .. After spinning out debris at 18,800 g for 15 min at 4°C, the supernatant was analyzed for the presence of mononucleosomes by extracting DNA and resolving it on a 2% agarose gel stained with ethidium bromide.

Clear Native PAGE:

Article Title: CAL1 is the Drosophila CENP-A assembly factor
Article Snippet: The nucleosomes were analyzed by 6% native PAGE and stained by SYBR gold; the incorporation of H2A–H2B was confirmed by fluorescence imaging of the gel for Alexa Fluor 488. .. MNase digestion of nucleosomes was conducted by incubating nucleosomes (∼60 nM) with 2,000 U/ml MNase (New England Biolabs, Inc.) for 30–120 s. After the reactions were stopped by addition of 20 mM EDTA, the samples were treated with 2% SDS and 0.4 mg/ml proteinase K for 30 min at 55°C.

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