Mnase

Manufactured by New England Biolabs

Sourced in United States

MNase is a micrococcal nuclease, an enzyme that cleaves DNA at the linker regions between nucleosomes. It is commonly used in chromatin biology research to study chromatin structure and dynamics.

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Lab products found in correlation

Proteinase k, by New England Biolabs (5 mentions) Proteinase k, by Thermo Fisher Scientific (5 mentions) Mnase buffer, by New England Biolabs (4 mentions) Qiaquick gel extraction kit, by Qiagen (4 mentions) Nebnext ultra 2 dna library prep kit, by New England Biolabs (3 mentions) Dnase 1, by New England Biolabs (3 mentions) Qiaquick pcr purification kit, by Qiagen (3 mentions) Complete protease inhibitor cocktail, by Roche (3 mentions) Bioruptor, by Diagenode (3 mentions) Rnase a, by Thermo Fisher Scientific (3 mentions) Sytox green, by Thermo Fisher Scientific (3 mentions) Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (2 mentions) Clc genomics workbench 20, by Qiagen (2 mentions) Proteinase k, by Roche (2 mentions) Rnase inhibitor, by New England Biolabs (2 mentions) Sybr green, by Meridian Bioscience (2 mentions) Quantstudio v1, by Thermo Fisher Scientific (2 mentions) Viia7, by Thermo Fisher Scientific (2 mentions) Dnase 1, by Promega (2 mentions) Ez view red anti flag m2 affinity gel beads, by Merck Group (2 mentions)

Close spelling variants of this product

MNase buffer (8 citations) Micrococcal nuclease (MNase) (16 citations) MNase reaction buffer (2 citations) MNase enzyme (2 citations)

118 protocols using mnase

Chromatin Accessibility Mapping by MNase-qPCR

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Cells (8 × 10⁴) were plated on a collagen-coated 35 mm dish. Following treatments, cells were permeabilized with CSK100 for 10 min at RT, gently washed with MNase buffer (50 mM Tris-HCl pH 8.0, 5 mM NaCl, 5 mM CaCl₂), and then treated with the 75 gelU/ml of MNase (New England Biolab, Cat# M0247S) in MNase buffer for 5 ~ 20 min at 25 °C. The MNase-treated cells were lysed in 250 μl of TES buffer (10 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.5% SDS). The cell lysates were sequentially treated with 1 mg/ml RNaseA (NipponGene, Cat# 318-06391) for 45 min and with 200 μg/ml Proteinase K (QIAGEN, Cat# 19133) for 90 min at 50 °C, after which an equal volume of phenol/chloroform (NipponGene, Cat# 311-90151) was added. The insoluble fraction was pelleted at 20,000 x g, the supernatant was mixed with 0.5 M of NaCl, and an EtOH wash was performed. The extracted DNA was resuspended in TE buffer (pH 8.0), resolved by 1.2% agarose gel (Fast Gene, Cat# NE-AG02) electrophoresis at 1 μg per lane, and stained with EtBr (NipponGene, Cat# 315-90051). Images were captured with a LAS 3000 luminescent image analyzer (FujiFilm) for intensity analysis with ImageJ.

Igarashi T., Mazevet M., Yasuhara T., Yano K., Mochizuki A., Nishino M., Yoshida T., Yoshida Y., Takamatsu N., Yoshimi A., Shiraishi K., Horinouchi H., Kohno T., Hamamoto R., Adachi J., Zou L, & Shiotani B. (2023). An ATR-PrimPol pathway confers tolerance to oncogenic KRAS-induced and heterochromatin-associated replication stress. Nature Communications, 14, 4991.

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Chromatin-bound Nuclear Protein Extraction

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For chromatin bound nuclear extracts, GM637 HF cells were washed twice with ice-cold PBS buffer. Cells were lysed with CSK (Cytoskeleton) buffer (10mM Hepes pH 6.8, 100mM NaCl, 300mM sucrose, 3mM MgCl2, 1mM EGTA, 0.1% Triton X-100, e-complete protease inhibitors) and chromatin extracts were crosslinked with 1% formalin (Sigma-Aldrich) in PBS buffer for 10min at room temperature followed by 125mM glycine addition. Cell pellets were resuspended in micrococal nuclease (MNase) buffer containing 2,000 units/mL of MNase (NEB). Extracts were incubated at room temperature for 10min and then diluted with an equal volume of 2X immunoprecipitation buffer (300mM NaCl, 50mM Tris-HCl pH7.5, 2mM EDTA, 0.5% TritonX100, 10% glycerol, phosphatase inhibitor and protease inhibitors). The extracts were solubilized by sonication (4× 10sec with 30sec interval) and isolated by centrifugation at 17,000g for 15min at 4°C. 2mg of chromatin extracts were mixed with 40 µL of Flag agarose beads (Sigma-Aldrich) overnight at 4°C. Flag agarose beads were washed with IP buffer twice and bound proteins were eluted in 2x sample buffer (20% glycerol, 125mM Tris-HCl pH6.8, 5% β-mercaptoethanol, 50mM DTT, 0.05% bromophenol blue). PCNA, Rad18, Flag ab were used for western blot analysis.

Yoon J.H., McArthur M.J., Park J., Basu D., Wakamiya M., Prakash L, & Prakash S. (2019). Error-prone replication through UV lesions by DNA polymerase θ protects against skin cancers. Cell, 176(6), 1295-1309.e15.

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MNase Assay for Chromatin Analysis

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The MNase assay was performed according to Infante et al.^{71 (link)} starting from BY4742 cells cultured in Ni(S-tcitr)₂ (15 µM)- or vehicle-containing YPD up to a density of 6–7 × 10⁶ cells/mL. After crosslinking with 1% (v/v) formaldehyde and spheroplast production by zymolyase treatment, DNA was digested with 1–10 units of MNase (New England Biolabs) for 30 min at 37 °C. The experiments were performed in triplicate from independent cultures.

Baruffini E., Ruotolo R., Bisceglie F., Montalbano S., Ottonello S., Pelosi G., Buschini A, & Lodi T. (2020). Mechanistic insights on the mode of action of an antiproliferative thiosemicarbazone-nickel complex revealed by an integrated chemogenomic profiling study. Scientific Reports, 10, 10524.

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MNase Digestion of Olfactory Nuclei

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For MNase digestion, olfactory neuroepithelia were harvested from Mecp2 WT
littermate adult mice and resuspended in PIPES buffer (5 mM PIPES,
85 mM KCl, 0.5% NP-40, pH 8.0) at 4 °C. After
disruption with a dounce homogenizer, nuclei were collected by centrifugation.
Collected nuclei were washed once, resuspended in the MNase buffer and digested
with 0.5 units of MNase (New England Biolabs, Ipswich, MA, USA) per microlitre
volume for 15 min at 37 °C. MNase digestion was stopped by
putting the samples on ice and adding EDTA to a concentration of 10 mM.
After digestion with
0.1 μg μl⁻¹ RNase A
(Fermentas, Pittsburgh, PA, USA), DNAs were purified with DNA Purification
Magnetic Beads (Life Technologies, Grand Island, NY, USA), and pellets were
dissolved in H₂O. DNA fragments corresponding to mononucleosomes
(about 150 bp) were confirmed by using 2100 Bioanalyzer (Agilent
Technologies Santa Clara, CA, USA). MNase-seq libraries were prepared as
described (Bioo Scientific, Austin, TX, USA) using 5140-01 NEXTflex DNA
Sequencing kit and 514101 NEXTflex DNA Barcodes-6. High throughput sequencing
was done with Illumina Hi-Seq 2000.

Rube H.T., Lee W., Hejna M., Chen H., Yasui D.H., Hess J.F., LaSalle J.M., Song J.S, & Gong Q. (2016). Sequence features accurately predict genome-wide MeCP2 binding in vivo. Nature Communications, 7, 11025.

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Nucleosome Digestion Assay Protocol

Cited 1 time

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The assay was performed as previously described (33 (link)). In brief, cell nuclei were isolated using hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, and 1 mM dithiothreitol (DTT)) and 0.1% Triton X-100. After washing in hypotonic buffer without Triton X-100, the cell nuclei were resuspended in reaction buffer (15 mM Tris–HCl, pH 7.4, 60 mM KCl, 0.25 M sucrose, 1 mM CaCl₂, and 0.5 mM DTT). The digestion was carried out with MNase (New England BioLabs) at 4 U MNase per 200 μl of reaction buffer at 25°C for the indicated period of time. The reaction was terminated by adding an equal volume of 2 × TNESK buffer (20 mM Tris–HCl, pH 7.4, 0.2 M NaCl, 2 mM EDTA and 2% SDS) with freshly added proteinase K (0.2 mg/ml). The samples were then incubated overnight at 37°C, and genomic DNA was purified and separated by agarose gel electrophoresis.

Shen T., Ji F., Wang Y., Lei X., Zhang D, & Jiao J. (2017). Brain-specific deletion of histone variant H2A.z results in cortical neurogenesis defects and neurodevelopmental disorder. Nucleic Acids Research, 46(5), 2290-2307.

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Isolation and Analysis of Mononucleosomes

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Cells were collected with trypsin, quantified, and centrifuged. The pellets were then lysed in 1 ml of NP-40 lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.5% NP-40, 0.15 mM spermine, 0.5 mM spermidine) per 10⁶ cells on ice for 5 min. After centrifugation, the pellets were then resuspended in MNase digestion buffer (10 mM Tris-HCl pH 7.4, 15 mM NaCl, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine), centrifuged, and resuspended again in the same buffer supplemented with 1 mM CaCl₂ and 120 units of MNase (New England Biolabs). Samples were incubated 30 min at 37 °C. Digestion was stopped by adding an equal volume of one part of STOP buffer (100 mM EDTA, 10 mM EGTA pH 7.5) and three parts of digestion buffer. Nucleosomes were removed by proteinase K digestion. Monosomes were gel extracted using the Gel extraction kit (Qiagen) and DNA was diluted to 5 ng/μl prior to quantitative PCR. The signal of the PCR was normalized to total sonicated DNA (Input) prepared as described in the ChIP section. Four replicates of each sample were processed, and the experiment was performed once.

Bandiera R., Wagner R.E., Britto-Borges T., Dieterich C., Dietmann S., Bornelöv S, & Frye M. (2021). RN7SK small nuclear RNA controls bidirectional transcription of highly expressed gene pairs in skin. Nature Communications, 12, 5864.

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Mapping H1 Binding on Chromatin

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H1 binding to intact and tailless chromatin was analyzed by micrococcal nuclease digestion. Chromatin templates (150 ng) were digested by MNase (New England Biolabs) at 3.5 units/ml during 1 min at 37 °C in MNase buffer. The reaction was stopped by MNase stop solution (300 mM NaAc (ph=5.3), 20 mM EDTA (pH=8.0)). DNA was purified and radioactively labeled by T4 PNK (New England Biolabs). Labeled DNA was purified and analyzed by 8% PAG (39:1). The gel was dried, exposed and scanned (BioRad, USA).

Nizovtseva E.V., Polikanov Y.S., Kulaeva O.I., Clauvelin N., Postnikov Y.V., Olson W.K, & Studitsky V.M. (2019). OPPOSITE EFFECTS OF HISTONE H1 AND HMGN5 PROTEIN ON DISTANT INTERACTIONS IN CHROMATIN. Molekuliarnaia biologiia, 53(6), 1038-1048.

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MNase Digestion and Sequencing Protocol

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SV129 M (100k) and RS (200k) cells were digested with MNase as previously described (Brind’Amour et al., 2015 (link)) with slight modifications. Frozen cell aliquots were first lysed in nuclear isolation buffer (Sigma) with protease inhibitors (Roche). MNase digestions (0U, 0.5U, 1U, or 2U, MNase (NEB)) were subsequently carried out in MNase Buffer (NEB) supplemented with 200mM DTT for 7.5min at 37°C. Reactions were stopped by adding EDTA. The resulting digested DNA was phenol:chloroform purified, quantified (Qubit), and tested for quality and correct nucleosomal size distribution with Bioanalzyer DNA 1000 analysis (Agilent). Sequencing libraries were generated from 37.5ng of DNA with the NEB Next Ultra II DNA Library Prep Kit for Illumina (5 amplification cycles) as described by manufacture protocol. Libraries were sequenced on the Illumina NextSeq with paired-end sequencing for 75 cycles.

Luense L.J., Donahue G., Lin-Shiao E., Rangel R., Weller A.H., Bartolomei M.S, & Berger S.L. (2019). Gcn5-mediated histone acetylation governs nucleosome dynamics in spermiogenesis. Developmental cell, 51(6), 745-758.e6.

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Preparing Nucleosomes from Cells

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For making nucleosomes without H2BK12Ac or H2BK120ub, H2B-Flag WT/K12R/K120R were transfected into cells. Then, cells were lysed by MNase lysis buffer (50 mM Tris–HCl [pH 7.5], 1 mM CaCl₂, 0.2% NP-40, Inhibitor Complex) and treated 50 U MNase (#M0247S, NEB) for making mono-nucleosomes. After MNase digestion, H2B-Flag containing mono-nucleosomes were pull down by Flag-M2 bead overnight, then these mono-nucleosomes were eluted by 3× Flag peptides (#F4799, Sigma).

Oh S., Boo K., Kim J., Baek S.A., Jeon Y., You J., Lee H., Choi H.J., Park D., Lee J.M, & Baek S.H. (2020). The chromatin-binding protein PHF6 functions as an E3 ubiquitin ligase of H2BK120 via H2BK12Ac recognition for activation of trophectodermal genes. Nucleic Acids Research, 48(16), 9037-9052.

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Co-Immunoprecipitation of Mononucleosome Complexes

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Co-IPs were conducted on mononucleosome fractions derived by Micrococcal Nuclease (MNase) treatment. Nuclei were prepared as above and resuspended in chromatin digestion buffer [50 mM Tris pH 7.4, 4 mM MgCl₂, 1 mM CaCl₂, EDTA-free protease inhibitor (Roche), 5 mM sodium butyrate] and incubated with MNase (20 KU, NEB) for 15 min at 37°C before the reaction was stopped by adding EDTA to a final concentration of 10 mM. The MNase fraction was derived by centrifugation at 10,000 rpm 5 min at 4°C. The MNase fraction was diluted 1/7 in RIPA buffer [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% IGEPAL CA-630, 1 mM EDTA, EDTA-free protease inhibitor (Roche)] and protein complexes were precipitated over night at 4°C with protein G agarose beads (GE Healthcare) and mouse anti-Ty1 or rat anti-HA antibodies. The complexes were washed five times in RIPA buffer and eluted using 2 × Laemmli buffer (125 mM Tris-HCl pH 6.8, 20% glycerol, 4% SDS, 0.005% bromophenol blue, 5% beta-mercaptoethanol). Co-IP extracts were analyzed by western blot or processed for Mass Spectrometry.

Quinn J.E., Jeninga M.D., Limm K., Pareek K., Meißgeier T., Bachmann A., Duffy M.F, & Petter M. (2022). The Putative Bromodomain Protein PfBDP7 of the Human Malaria Parasite Plasmodium Falciparum Cooperates With PfBDP1 in the Silencing of Variant Surface Antigen Expression. Frontiers in Cell and Developmental Biology, 10, 816558.

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