Purified cells were fixed with 1% formaldehyde (Sigma) at a concentration of approximately 106 cells/ml. Fixed cell preparations were sonicated using a Diagenode Bioruptor UCD-300 for 3x10 min (30 s on; 30 s off). 67 μl of chromatin (1 million cells) were incubated with 229 μl dilution buffer, 3 μl protease inhibitor cocktail and 0.5–1 μg of H3K4me3 antibody (Diagenode) and incubated overnight at 4°C with rotation. Protein A/G magnetic beads were washed in dilution buffer with 0.15% SDS and 0.1% BSA, added to the chromatin/antibody mix and rotated for 60 min at 4°C. Beads were washed with 400 μl buffer for 5 min at 4C with five rounds of washes. After washing chromatin was eluted using elution buffer for 20 min. Supernatant was collected, 8 ml 5M NaCl, 3 ml proteinase K were added and samples were incubated for 4 h at 65°C. Finally, samples were purified using QIAGEN; Qiaquick MinElute PCR purification Kit and eluted in 20 ml EB.
Protease inhibitor cocktail
Manufactured by Diagenode
Sourced in Belgium
The Protease Inhibitor Cocktail is a laboratory reagent designed to inhibit the activity of various proteases. It is formulated to provide a broad spectrum of protection against a wide range of proteolytic enzymes, preventing their degradation of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bioruptor plus, by Diagenode (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
H3k4me3 antibody, by Diagenode (1 mentions)
Bioruptor ucd 300, by Diagenode (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000 system, by GE Healthcare (1 mentions)
Picoruptor, by Diagenode (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Protease inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Chip grade protein g magnetic beads, by Cell Signaling Technology (1 mentions)
Simplechip plus sonication chromatin ip kit, by Cell Signaling Technology (1 mentions)
Luna universal qpcr master mix kit, by New England Biolabs (1 mentions)
Proteinase k, by Cell Signaling Technology (1 mentions)
Dna purification kit, by Cell Signaling Technology (1 mentions)
H3k27ac antibody, by Abcam (1 mentions)
Bioruptor pico sonicator, by Diagenode (1 mentions)
Pierce ripa buffer, by Thermo Fisher Scientific (1 mentions)
7 protocols using protease inhibitor cocktail
1
ChIP-seq protocol for H3K4me3
2
Western Blot Analysis of ETV6-RUNX1 Fusion
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Cells were pelleted, washed with PBS Buffer and lysed (30 min on ice with occasional vortexing) in RIPA Buffer (Sigma Aldrich) supplemented with 1× Protease Inhibitor Cocktail (Roche). Lysates were cleared by centrifugation at 4 °C for 15 min at 14,000 × g and total protein extracts were transferred to a new pre-chilled Eppendorf tube. For insoluble fraction extracts (ETV6-RUNX1) the remaining pellet was resuspended in 50 μl RIPA supplemented with 1× Protease Inhibitor Cocktail and sonicated for 3-5 min with Picoruptor® (Diagenode). Ten micrograms of proteins were denatured, run on precast 4–12% NuPAGE® Bis-Tris gels (Invitrogen) and transferred onto a PVDF membrane (Millipore). Protein membrane was blocked and incubated with primary antibody O/N at 4 °C. Following 1h incubation with secondary antibodies, membranes were developed using the ImageQuant LAS 4000 System (GE Healthcare). Antibodies used: RUNX1 (1:3000 dilution, Abcam, ab23980), ETV6: (1:1000, Sigma, HPA000264), CBFβ (1:1000 dilution, Abcam, ab33516), GAPDH (1:5000 dilution, 14C10, Cell Signaling #2128).
3
GST Pull-Down Assay with Modifications
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The GST pull-down assay was performed as previously described [10 (link)] with some modifications: Pellets from 50 mL of bacterial culture were sonicated at 4 °C using Bioruptor® Plus (Diagenode) in lysis buffer (1× PBS, 1 mM EDTA, 0.1% Triton X-100 (v/v), 1 mg/mL lysozyme) containing EDTA-free protease inhibitor cocktail (Roche). GST-pull down assays were performed at least in triplicate.
4
ChIP-seq protocol for H3K27ac profiling
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The SimpleChIP Plus Sonication Chromatin IP Kit (56383, Cell Signalling) was used, following manufacturer’s guidelines. In brief, after resuspension of the chromatin pellet in ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.0 plus 1 X protease inhibitor cocktail), samples were subjected to fragmentation using a Bioruptor pico sonicator (Diagenode) for 5 cycles (30 s ON and 30 s OFF). 5 μg of sonicated, cross-linked chromatin sample per condition was diluted in a 1:4 ratio in 1X ChIP buffer (Cell Signalling) plus 1X protease inhibitor cocktail (Cell Signalling) and subjected to immunoprecipitation with 2.5 μg H3K27ac antibody (Abcam) overnight at 4 °C with rotation. 30 μl of ChIP-Grade Protein G Magnetic Beads (Cell Signalling) were then added to each IP reaction and incubated for 2 h at 4 °C with rotation, prior to a series of low and high salt washes and elution of antibody-protein-DNA complexes in 1X ChIP Elution Buffer. Enriched chromatin samples were then incubated at 65 °C for 2 h with 40 μg Proteinase K (Cell Signalling) to reverse cross-links prior to purification of DNA using the DNA Purification Kit (Cell Signalling) as per manufacturer’s guidelines. DNA was eluted in 50 μL of elution buffer prior to subsequent qPCR using the Luna Universal qPCR Master Mix kit (New England BioLabs).
5
Protein Extraction from HTLV-1 Cells
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Total proteins were extracted from uncultivated PBMCs or human T cell lines using Pierce RIPA Buffer (Thermo Fisher Scientific, Yokohama, Japan) with protease inhibitor cocktail (Thermo Fisher Scientific). Briefly, 1 × 107 HTLV-1-infected and uninfected cells were washed three times with PBS, resuspended in Pierce RIPA Buffer with protease inhibitor cocktail, and then sonicated on ice using a Bioruptor® sonicator (Diagenode, Liège, Belgium) according to the manufacturer’s protocol. After centrifugation (14,000×g, 4 °C, 15 min), the supernatants were collected for western blotting and sandwich ELISA.
6
Chromatin Immunoprecipitation from MM2d Cells
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MM2d cells were harvested 4 days after subculture and fixed using ice-cold 1% formaldehyde in PBS and applying vacuum infiltration (three rounds of 6 min on/4 min off). The cross-linking was stopped by the addition of 0.125 M glycine, infiltrating for another 5 min. The grinded material was resuspended in Extraction Buffer (0.25 M sucrose, 10 mM Tris–HCl, pH 8.0, 10 mM MgCl2, 1% Triton X-100, 1 mM PMSF and protease inhibitor cocktail for plant cell extracts (Sigma)). Nuclei were pelleted by centrifugation, resuspended in Lysis Buffer (50 mM Tris–HCl, pH 8.0, 10 mM EDTA, 1% SDS, 1 mM PMSF and protease inhibitor cocktail) and disrupted by sonication in a Bioruptor Plus (Diagenode) for 30–45 cycles of 30 s on and 30 s off, at high power mode. One μg of soluble chromatin was employed per ChIP reaction, using the following antibodies: anti-H3K9me2 (Abcam ab1220, 3 μg), anti-H3K27me1 (Millipore 07-448, 1 μg), anti-total H3 (Abcam ab1791, 2 μg), or anti-rat IgG (Abcam ab6703, 2 μg) as a negative control. Immune complexes were recovered with 50 μl of protein G agarose beads (SCBT) and washed and eluted essentially as described (28 (link)).
7
Chromatin Immunoprecipitation Protocol
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Cells were cross-linked with 1% formaldehyde in PBS for 10 min at room temperature followed by quenching with 125 mM glycine. Cells were lysed in lysis buffer 1 [50 mM Hepes-KOH (pH 7.5), 140 mM NaCl, 1 mM 0.5 M EDTA, 10% glycerol, 0.5% IGEPAL CA630, and 0.25% Triton X-100] containing protease inhibitor cocktail (Sigma-Aldrich, P8340) for 30 min at 4°C and washed in lysis buffer 2 [10 mM tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA]. The nuclear pellet was resuspended in IP buffer [50 mM tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 20% glycerol, 0.1% NP-40, and 1.5 mM MgCl2] containing protease inhibitor cocktail, followed by mild sonication using Diagenode Bioruptor. Insoluble proteins were separated by spinning for 30 min at 13,000 rpm at 4°C. The extract was incubated with anti-FLAG M2 agarose beads (Sigma-Aldrich, A2220) and benzonase (Merck, 71205-3) overnight on a rotating wheel at 4°C. The beads were washed in IP buffer for 5 min at 4°C with rotating. The wash step was repeated five times. The beads were eluted by boiling in SDS sample buffer for 5 min.
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