2 µg of His-C-clamp and 2 µg of either GST or GST-HMG were incubated for 1 hour at 4°C with rotation in 20 mM Tris-HCl (pH 7.62), 150 mM NaCl and 1% Triton X-100 binding buffer. The mixture was then incubated with Glutathione Sepharose beads (GE Healthcare) for an additional 2 hours at 4°C with rotation. After 4–5 washes with binding buffer, the sample was treated with hot SDS buffer and loaded into a gel. His-C-clamp was detected using mouse anti-His antibody (1∶3000, GE Healthcare). Micrococcal nuclease treatment was carried out as described previously [56] (link), using 6.6 units of Micrococcal nuclease (Sigma) for a 200 µL reaction volume. In addition, the binding buffer used for this treatment contained 5 mM CaCl2. OPA treatment was carried out by incubating His-C-clamp in 0.363 µM OPA for 20 minutes at room temperature. GST or GST-HMG was then added and the mixture was incubated for 1 hour at 4°C with rotation prior to GST pulldown. In the negative controls for both treatments, nuclease free water was used instead of Micrococcal nuclease or OPA.
Micrococcal nuclease
Manufactured by Merck Group
Sourced in United States, Germany
Micrococcal nuclease is a laboratory enzyme used to cleave DNA and RNA. It is a calcium-dependent endonuclease that non-specifically hydrolyzes the phosphodiester bonds in nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Instant imager, by Hewlett-Packard (6 mentions)
Calf spleen phosphodiesterase, by MP Biomedicals (5 mentions)
Hp601nd, by Hartmann Analytic (2 mentions)
Varioskan flash, by Thermo Fisher Scientific (2 mentions)
Vivaspin devices, by GE Healthcare (2 mentions)
Model 491 preparative cell, by Bio-Rad (2 mentions)
H3k4me3, by Merck Group (2 mentions)
Calf thymus dna, by Merck Group (2 mentions)
Rnase a, by Merck Group (2 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (2 mentions)
Spleen phosphodiesterase, by Merck Group (2 mentions)
Phosphatase inhibitor cocktails 1 and 2, by Merck Group (2 mentions)
Omnicleave endonuclease, by Illumina (2 mentions)
Rnase, by Merck Group (2 mentions)
Dnase 1, by Roche (2 mentions)
Omnicleave, by Roche (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Benzonase, by Merck Group (2 mentions)
Nuclease p1, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
68 protocols using micrococcal nuclease
1
Probing Protein-Protein Interactions
2
Quantifying DNA Adducts via 32P-Postlabelling
3
Neutrophil Extracellular Trap (NET) Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NET production was performed as previously described (Von Köckritz-Blickwede et al., 2010 (link)). Briefly, 2 × 105 neutrophils in HBSS+/+ were seeded in a 96-wells plates, and 25 nM PMA (positive control) or B. anthracis at approximately an MOI of 5 were added to the wells. The plate was spun down at 290 × g for 5 min and incubated at 37°C, 5% CO2 for 4 h. Next, 500 U/ml of micrococcal nuclease (Sigma) was added to the wells and incubated at room temperature to digest the NETs. Then 5 mM EDTA was added to stop the activity of the micrococcal nuclease and cells removed by centrifugation. Supernatant was collected and incubated with Quanti-iT PicoGreen for 5 min at room temperature. Fluorescence signal was measured at excitation 480 nm/emission 520 nm on a SpectraMax Gemini EM fluorescence reader. To visualize NET structures, 5 × 104 neutrophils in HBSS+/+ were seeded in borosilicate 8-well chamber slides (Nunc). Next 25 nM PMA (positive control) or 25 × 104 CFU of B. anthracis in HBSS+/+ were added to each chamber. Slides were then incubated at 37°C, 5% CO2 for 4 h, after which 4% paraformaldehyde was added and the content of the chambers fixed for 30 min at room temperature. Chambers were then washed three times with PBS, and NET structures stained with 5 μM SYTOX Green (Invitrogen). NETs were imaged on a Zeiss Observer D1 inverted fluorescent microscope.
4
DNA Adduct Measurement by 32P-Postlabelling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was isolated from cells or tissue by a standard phenol/chloroform extraction method and stored at −20 °C. DNA adducts were measured in each DNA sample using the butanol enrichment version of the 32P-postlabelling method as described previously [32] (link), [35] (link) with minor modifications. Briefly, DNA samples (4 μg) were digested with micrococcal nuclease (120 mU; Sigma, #N3755) and calf spleen phosphodiesterase (40 mU; Calbiochem, #524711), enriched and labelled with 50 μCi [γ-32P]ATP (Hartmann Analytic #HP601ND) as reported. 3-NBA-derived DNA adducts were identified as reported [11] (link), [12] (link), [13] (link).
5
Quantifying Cell-free DNA Release
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After co-incubation for 2 h, the culture medium described in NET formation was prepared for dsDNA determination. 0.1 M CaCl2 and 50 U/ml micrococcal nuclease (Sigma-Aldrich, USA) were added for 10 min for DNA fragmentation and digestion. Next, 0.5 M ethylenediaminetetraacetic acid (EDTA) was added to stop the reaction. Then, the supernatants were collected and incubated with Quant-iTTM PicoGreen® reagent (Invitrogen, UK) in a ratio of 1:1 to determine the cell-free DNA, according to the manufacturer’s instructions. After incubation for 5 min in the dark, the DNA mixture was measured at an excitation wavelength of 480 nm and emission wavelength of 530 nm by spectrofluorometer (Varioskan Flash, Thermo fisher scientific, Finland).
6
DNA Manipulation and Cell Culture Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Acridine orange, chloramphenicol, 4′,6-diamidino-2-phenylindole (DAPI), DNA from salmon testes, HEPES buffer, LB EZ Mix agar, and zidovudine were from Sigma-Aldrich (now Millipore Sigma). DNase I, from the bovine pancreas, and micrococcal nuclease, were also both from Sigma-Aldrich. Aztreonam and bleomycin were from Cayman Chem, Ann Arbor, MI. Agarose for DNA gels was SeaKem LE agarose from Lonza Co., Rockland, ME. Antibiotic MIC Strips were from Liofilchem, Waltham, MA.
DMEM/F12 powder was from the Gibco Division of Thermo-Fisher, Grand Island, NY, USA. The composition of this medium is available athttps://www.thermofisher.com/us/en/home/technical-resources/media-formulation.329.html , accessed on 16 March 2023. The DMEM/F-12 was supplemented with 18 mM NaHCO3 and 25 mM of additional HEPES, pH 7.4. Sybr Safe DNA stain, 100 bp DNA ladders, and 10 X loading dye (“Blue Juice”) were from the Invitrogen Division of Thermo-Fisher.
DMEM/F12 powder was from the Gibco Division of Thermo-Fisher, Grand Island, NY, USA. The composition of this medium is available at
7
Isolation and Characterization of Chromatin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatin and nucleosomes were prepared under sterile conditions from calf thymus as previously described (6 (link), 25 (link)). Briefly, nuclei were isolated and then digested by micrococcal nuclease (Sigma-Aldrich). The reaction was stopped by EDTA and centrifuged. The pellet was harvested and nuclei were lyzed. After centrifugation, the supernatant containing chromatin was collected. This fraction is composed of chromatin fragments of different sizes, including high molecular weight complexes. When used in cell culture, the lysis buffer served as a negative control. In other experiments, mono-nucleosomes were used. In that case, chromatin was further purified by ultracentrifugation on 5–29% sucrose gradients. As a negative control in cell culture, the purification buffer was used, i.e., an empty sucrose gradient loaded with lysis buffer only. All chromatin fractions were analyzed by agarose gel electrophoresis (1.5%) and SDS-PAGE (18%). Of note, free self DNA is not strongly immunogenic and histones are 100% conserved in human and calf.
8
Isolation and Purification of Native Mononucleosomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Native human mononucleosomes were prepared according to established protocols28 with minor modifications. HeLa cells were lysed in 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM phenylmethanesulfonylfluoride (PMSF), 10 mM Hepes-NaOH, pH 7.9using a 40-ml Dounce homogenizer (Wheaton). Nuclei, harvested by gentle centrifugation, weredigested with 0.5 U/ml micrococcal nuclease (Sigma-Aldrich) in 4 volumes 0.34 M sucrose, 3 mM CaCl2, 60 mM KCl, 0.5 mM PMSF and 50 mM Tris-HCl, pH 7.5for 10 min at 37 °C. The reaction was stopped with 50 mM EDTA, and the nucleosomes, extracted by addition of 0.5 M NaCl, were dialyzed overnight at 4 °C against 650 mM NaCl, 2 mM EDTA, 1 mM β-mercaptoethanol, and 20 mM Hepes, pH 7.4. Nucleosomes were isolated by size exclusion chromatography through a Superdex-200 column operated in 650 mM NaCl, 1 mM EDTA and 20 mM Tris-HCl, pH 7.5.For the use in strand transfer assays (Fig. 1a ), nativenucleosomes, purified by size exclusion chromatography and dialyzed overnight against 100 mM NaCl, 1 mM EDTA and 20 mM Tris-HCl, pH 7.5, were separated by native 8% PAGE in a Model-491 preparative cell (Bio-Rad Laboratories). Nucleosomes were concentrated to 3 mg/ml using Vivaspin devices (GE Healthcare) and stored on ice.
9
Chromatin Immunoprecipitation of FLAG-HRP2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
293T cells were transfected with FLAG-tagged HRP2 or mutated HRP2 (W21A). 24 hours later, nuclei were prepared and resuspended in 10 mM Tris–HCl pH 7.5, containing 10 mM NaCl, 3 mM MgCl2, 3 mM CaCl2, 0.1 mM PMSF and chromatin was released by digestion with micrococcal nuclease (Sigma) at 37°C for 10 min. Digestion was optimized to produce primarily mono-nucleosomes. Digestion was stopped by addition of 20 mM EDTA. Nuclear debris was removed by centrifugation and the soluble chromatin was incubated with anti-FLAG M2 affinity gel at 4°C overnight. After washing four times, SDS loading buffer was added and boiled for 10 min. The supernatant were subjected to immunoblotting. Antibodies used were FLAG-HRP (Sigma, F7425), Histone H3 (Abcam, ab1791), H3K36me2 (Abcam, ab9049), H3K36me3 (Abcam, ab9050), H3K4me3 (Millipore, 05-745), H3K9me3 (Millipore, 07-442) and H3K27me3 (Abcam, ab6002).
10
Neutrophil NETosis Modulates Tumor Cell Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
1 × 105 neutrophils were treated with CAF CMed diluted 1:1 in complete EC media (Cell Biologics) with or without Cl-amidine and incubated for 3 h to generate NETs. The media was then harvested from the NETting or NET inhibited neutrophils. PBS supplemented with 1U/ml micrococcal nuclease (Sigma-Aldrich) was then added to the neutrophils for 10 min at 37 °C and 5% CO2 to detach the NETs. The enzyme was inactivated with 0.5 mM EDTA. PBS containing the NETs was then harvested for xCELLigence assays.
Pancreatic CAF or tumor cells were seeded onto 16 well E-plates (ACEA Biologics) at a density of 5 × 103 cells/well. Cells were allowed to adhere for 1 h. The media was then replaced with the NETting or NET inhibited neutrophil CMed. Plates were placed into an xCELLigence RTCA MP Real-Time Cell Analyzer (ACEA Biologics). Recordings of the impedance, correlating to cell proliferation, were then taken over a 48 h period. The background was then subtracted from the appropriate wells.
Pancreatic CAF or tumor cells were seeded onto 16 well E-plates (ACEA Biologics) at a density of 5 × 103 cells/well. Cells were allowed to adhere for 1 h. The media was then replaced with the NETting or NET inhibited neutrophil CMed. Plates were placed into an xCELLigence RTCA MP Real-Time Cell Analyzer (ACEA Biologics). Recordings of the impedance, correlating to cell proliferation, were then taken over a 48 h period. The background was then subtracted from the appropriate wells.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!