Cells were crosslinked in 1% formaldehyde for 10 min at 37 °C with constant stirring, followed by quenching with 125 mM glycine for 5 min at room temperature. Cells were rinsed with 1X PBS and lysed, and chromatin was sheared using a Branson sonicator to a DNA fragment range of 200–1000 base pairs. Chromatin was incubated with antibody at 4 °C overnight with constant rotation. Co-immunoprecipitation of antibody-protein complexes was performed using Protein A or Protein G Dynabeads for 1 h at 4 °C. ChIP libraries were completed using previously reported method51 (link). In brief, immunoprecipitated DNA was end repaired using the End-IT DNA End-Repair Kit (Epicentre), extended using Klenow fragment (3’−5’ exo) (NEB), and ligated to sequencing adapter oligos (Illumina). Each library was PCR-amplified using PFU Ultra II Hotstart Master Mix (Agilent), and a size range of 300–600 base pairs selected for sequencing. Immunoprecipitation was carried out using following antibodies: OCT4 (Santa Cruz, sc-8628x; 5 ug per 106 cells), SOX2 (Santa Cruz, sc-17319; 5 ug per 106 cells), H3K4me2 (Millipore, 07–030; 5 ul per 106 cells), H3K27ac (Diagenode, C15410196; 1 ug per 106 cells). Libraries were sequenced on the Illumina Hiseq 2000.
Sonicator
Manufactured by Emerson
Sourced in United States
The Sonicator is a lab equipment product designed to generate high-frequency sound waves. It is used to disrupt and homogenize samples, as well as to extract and separate components from various materials. The Sonicator operates by converting electrical energy into mechanical vibrations, which are then used to agitate the sample and facilitate various laboratory processes.
Automatically generated - may contain errors
Lab products found in correlation
Cfx96 real time system, by Bio-Rad (3 mentions)
Sybr green supermix, by Bio-Rad (3 mentions)
Phosstop, by Roche (2 mentions)
2 mm microtip, by Emerson (2 mentions)
Chip kit, by Merck Group (2 mentions)
Trimethyl h3 k27, by Abcam (2 mentions)
Protein g a beads, by Thermo Fisher Scientific (2 mentions)
T per buffer, by Thermo Fisher Scientific (2 mentions)
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Epiquik tissue chromatin immunoprecipitation kit, by Epigentek (2 mentions)
Homogenizing buffer, by Epigentek (2 mentions)
Nuclear lysis buffer, by Epigentek (2 mentions)
Anti smp30 sc377184, by Santa Cruz Biotechnology (2 mentions)
Antibody diluted buffer, by Emerson (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Sequencing adapter oligos, by Illumina (1 mentions)
End it dna end repair kit, by Illumina (1 mentions)
Pfu ultra 2 hotstart master mix, by Agilent Technologies (1 mentions)
H3k4me2, by Merck Group (1 mentions)
64 protocols using sonicator
1
ChIP-Seq Protocol for Pluripotency Factors
2
Cytokine Profiling of Frozen Tumors
3
JQ1 Dose-Dependent Western Blot Analysis
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For western blot analysis, 250 000 cells per ml of BL, AML, MM and
Eμ-Myc lymphoma cells were cultured in a total volume of 20 ml.
Twenty-four hours after plating, different concentrations of JQ1 (0, 50, 100, 250
and 500 nm ) were added to the culture for either 6 or
24 h. Cells were collected, washed once in PBS and lysed for 10 min on ice
in an adequate volume of lysis buffer (20 mm HEPES pH 7.5,
100 mm NaCl, 5 mM EDTA, 10%
glycerol, 1% Triton X-100) supplemented with MINI-complete Protease
Inhibitor Cocktail Tablets (Roche, Indianapolis, IN, USA) and phosphatase
inhibition (0.4 mm ortovanadate, 10 mm NaF).
The cell lysate was sonicated with Branson sonicator and cleared by centrifugation
at full speed at 4 °C. Proteins were quantified by Bradford assay.
Proteins (20–30 μg) were boiled at 95 °C with Laemmli
sample buffer and loaded on Mini-PROTEAN TGX Gel (Bio-Rad, Hercules, CA, USA).
Trans-Blot Turbo Transfer System (Bio-Rad) was used to transfer proteins to
Trans-Blot Turbo Nitrocellulose Transfer Packs (Bio-Rad). Blocking was performed
with Tris-buffered saline (TBS)+5% of non-fat milk or with
TBS+5% of bovine serum albumin. Primary antibody was incubated
overnight at 4 °C, whereas secondary antibody was incubated for
1 h at room temperature. The western blots were developed with ECL
(Amsharm) using the ChemiDoc System (Bio-Rad).
Eμ-Myc lymphoma cells were cultured in a total volume of 20 ml.
Twenty-four hours after plating, different concentrations of JQ1 (0, 50, 100, 250
and 500 n
24 h. Cells were collected, washed once in PBS and lysed for 10 min on ice
in an adequate volume of lysis buffer (20 m
100 m
glycerol, 1% Triton X-100) supplemented with MINI-complete Protease
Inhibitor Cocktail Tablets (Roche, Indianapolis, IN, USA) and phosphatase
inhibition (0.4 m
The cell lysate was sonicated with Branson sonicator and cleared by centrifugation
at full speed at 4 °C. Proteins were quantified by Bradford assay.
Proteins (20–30 μg) were boiled at 95 °C with Laemmli
sample buffer and loaded on Mini-PROTEAN TGX Gel (Bio-Rad, Hercules, CA, USA).
Trans-Blot Turbo Transfer System (Bio-Rad) was used to transfer proteins to
Trans-Blot Turbo Nitrocellulose Transfer Packs (Bio-Rad). Blocking was performed
with Tris-buffered saline (TBS)+5% of non-fat milk or with
TBS+5% of bovine serum albumin. Primary antibody was incubated
overnight at 4 °C, whereas secondary antibody was incubated for
1 h at room temperature. The western blots were developed with ECL
(Amsharm) using the ChemiDoc System (Bio-Rad).
4
Preparation of Lipid Vesicles with GM1 and GD1a
5
Quantitative Proteomics of HeLa Cells
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HeLa cells were lysed in a buffer consisting of 0.1 M Tris-HCl pH 8.0, 0.1 M DTT and 4% SDS at 95°C for 5 min. After chilling to room temperature, the lysates were sonicated using a Branson-type sonicator and then clarified by centrifugation at 16,000 g for 10 min. Protein content was determined by comparison to a tryptophan protein standard using a spectrophotometric method, with excitation wavelength 280 nm and emission wavelength 350 nm. A heavy SILAC standard was prepared by mixing heavy HeLa cells under a variety of conditions in order to cover the proteome of stressed and unstressed cells. Specifically, we mixed equal amounts of lysates from untreated and stressor-treated heavy HeLa cells to obtain a master mix. For each sample, 100 μg of light cell lysate was mixed with 100 μg of heavy master mix and further processed.
6
ChIP Assay Protocol with Antibody Immunoprecipitation
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ChIP assays were performed with a ChIP assay kit (Millipore, NY) by following the protocol provided by the manufacturer with slight modifications as previously described [51 (link)]. Briefly, 5 million cells were fixed with 1% formaldehyde and then sonicated for 180 s (10 s on and 10 s off) on ice using a Branson sonicator with a 2-mm microtip at 40% output control and 90% duty cycle settings. The sonicated chromatin was immunoprecipitated with specific antibodies to CTCF, SUZ12, and dimethyl-H3-K27 (lysine 27 of histone H3)(Cell Signaling, MA). Anti-IgG was used as the ChIP control in parallel with testing samples. ChIP DNAs were quantitated by qPCR using target gene primers (Supplementary Table 2 ). For comparison, the ChIP data are presented as relative values by normalizing to PCR signals of input DNA (i.e. ratio of the ChIP over the input).
7
Chromatin Immunoprecipitation of Bcl6
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Cells were fixed, nuclei lysed, and chromatin sheared with a Branson sonicator. Immunoprecipitation was performed with the Millipore ChIP kit according to the manufacturer’s protocol. To precipitate DNA–protein complexes, anti-Bcl6 antibody was used and goat IgG was used as an isotype control. Percentage input was determined by removing an aliquot of sheared chromatin prior to immunoprecipitation and comparing amplification of this DNA with amplification of the precipitated chromatin. Putative Bcl6 binding sites in the klf2, klf4, sox2, c-myc, oct4, sirt1, hif-1α, tet2 loci were investigated to assay the relative enrichment by qPCR. Primers were listed in Table S1.
8
Hydrophylloquinone Stabilization by Methylation
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The stabilization of hydrophylloquinone was achieved by di-O-methylation as described (Sussmann et al., 2016 (link)). Briefly, 4x108 infected erythrocytes labeled with [3H]-phytyl-PP were lysed by ultrasonication in a Branson sonicator at 4°C with three pulses of 5 second duration with 10% potency and 10 second intervals between them. Then, 50 mg of potassium hydroxide and 60 μL of methyl iodide (CH3I) were added. The reaction was stirred overnight under a nitrogen atmosphere at room temperature in the dark. The reaction mixture was partitioned between water (2 mL) and dichloromethane (3 × 2 mL). The organic phase was evaporated under nitrogen stream, and the residue was submitted to RP-HPLC analysis and scintillation counting.
9
Amyloid-Beta Oligomer Preparation
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Lyophilized Aβ42 peptides stored at -80°C were allowed to equilibrate to room temperature prior to dilution to 1 mM with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). HFIP was evaporated in a vacuum centrifuge in order to form Aβ42 peptide films and films were then stored at -80°C. Prior to use, Aβ42 peptide films were diluted in dimethylsulphoxide (DMSO) to 1 mM and sonicated for 10 minutes in a Branson sonicator. Aβ42 peptides were then subsequently diluted to 100 μM in ice-cold F-12 cell culture media (phenol free red), vortexed immediately for 30 seconds, and incubated at 4°C for 24 hours in order to form Aβ42 oligomers.
10
ChIP-seq Protocol for Protein-DNA Interactions
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ChIP experiments were performed as previously described11 (link). Briefly, cell cultures were cross-linked in 1% formaldehyde for 30 min. Cells were then broken using a FastPrep (MP Biomedicals), and the chromatin fraction was sheared to 200–500 bp fragments using a Branson sonicator for nine cycles (10 s ON, 50 s OFF) at an amplitude of 20%. For immunoprecipitation (IP), 3–5 µg of anti-HA (16B12) or anti-Myc antibodies (9E11) were incubated overnight at 4 °C with the chromatin extracts and then coupled with 50 µl of protein-G-sepharose beads (GE17–0618–01, Sigma) during 4 h at 4 °C. ChIP DNA was quantified by fluorescence-based quantitative PCR using SYBR Green, as described for RT-qPCR analysis. Input (IN) samples were diluted 200-fold, while IP samples were diluted threefold. Relative occupancy levels were determined by dividing the IP by the IN value (IP/IN) for each amplicon. To determine the specificity of enrichment of the tagged protein, the corresponding untagged control samples were included in each ChIP experiment. All primer sequences are listed in Supplementary Table 5 .
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