For experiments shown in Figs. 2 , 3 , 5 , and 6 , desalting buffer (0.25 M sucrose, 10 mM Tris-HCl, and 0.05 mM CaCl2, pH 7.2) was prepared. Detached cells were rinsed with PBS and lysed in 0.5% NP-40 (Sigma) in desalting buffer containing protease-inhibitor cocktail set III (Calbiochem, San Diego, CA) (1:100). The sample was rotated overnight at 4 °C, and then the supernatants were obtained by centrifugation. Protein contents of the lysates were determined by the BCA protein- quantification reagent (Pierce, Waltham, MA). For experiments shown in Fig. 7 , protein lysates were prepared using CytoBuster protein-extraction buffer (Millipore, Burlington, MA) according to the manufacturer’s instructions.
Cytobuster protein extraction buffer
Manufactured by Merck Group
Sourced in Germany
CytoBuster protein-extraction buffer is a laboratory reagent designed to facilitate the extraction of proteins from cells or tissue samples. It is a proprietary buffer solution that aids in the disruption of cell membranes and the solubilization of proteins, enabling their subsequent analysis or purification.
Automatically generated - may contain errors
Lab products found in correlation
Np 40, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Nb600 308, by Santa Cruz Biotechnology (1 mentions)
Mab374, by Merck Group (1 mentions)
Mouse il 1 beta il 1f2 duoset elisa, by R&D Systems (1 mentions)
Duoset ancillary reagent kit 2, by R&D Systems (1 mentions)
Quick start bradford protein assay kit, by Bio-Rad (1 mentions)
Mouse tnf alpha duoset elisa, by R&D Systems (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Molecular probes dcfh da, by Thermo Fisher Scientific (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
4 protocols using cytobuster protein extraction buffer
1
Cell Lysis and Protein Extraction
2
Whole Cell Protein Extraction and Immunoblotting
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Whole bone marrow cells from different genotypes of mice, lymphoblastoid cell lines, and human cell lines were lysed using a cytobuster protein extraction buffer (Millipore) with protease inhibitor cocktail (GenDEPOT). Standard immunoblotting analysis was performed using anti-GFP (1:1,000, Novus, NB600–308), anti-DNMT3A antibody (1:1,000, Santa Cruz, H-295; 1:1,000, Abcam, ab16704), anti-DCAF8 antibody (1:500, Sigma-Aldrich, HPA027381), anti-DDB1 antibody (1:1,000, Bethyl Laboratories, A300–462A), and anti-GAPDH antibody (1:2,000, Millipore, MAB374).
3
Quantifying Macrophage Cytokine Secretion
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E. coli LPS-stimulated TNFα and IL-1β secretion from peritoneal macrophages isolated from WT or Spock1-Tg mice were measured using DuoSet Ancillary Reagent Kit2 (DY008; R&D Systems), mouse TNF-alpha DuoSet ELISA (DY410-05; R&D Systems) and mouse IL-1beta/IL-1F2 DuoSet ELISA (DY401-05; R&D Systems) according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Hercules, California, USA). For quantification, peritoneal macrophages after stimulation were collected using 100 µL of CytoBuster Protein Extraction Buffer (Merck) for protein extraction. Protein concentration was measured using the Quick Start Bradford Protein Assay Kit (Bio-Rad Laboratories) and 4× LeammLi Sample Buffer (Bio-Rad Laboratories) according to the manufacturer’s protocol.
4
Quantifying ROS in Zebrafish Larvae
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The production of ROS in zebrafish larvae was determined using membrane-permeable fluorescent dye 2’,7’-dichlorodihydrofluorescin diacetate (Molecular Probes DCFH-DA; Thermo Fisher Scientific, USA) according to the method of Zeng et al. [23 (link)] with a minor modification. Three replicates of twenty larvae that had been treated with 0.3, 1.3, 5, 20 and 80 ppb EZA for 3 days were homogenized in CytoBuster protein extraction buffer (Merck, Germany). And the homogenate was centrifuged at 15,000×g at 4 °C for 20 min. The supernatant was incubated for 30 min with DCFH-DA in PBS (pH7.4). The fluorescence intensity was measured using a microplate reader (SpectraMax M5, Molecular Devices) with excitation and emission at 485 nm and 530 nm, respectively. The ROS levels were estimated based on a DCF (dichlorofuorescein) standard curve, and expressed as an arbitrary unit (μg DCF /mg protein) and as a percentage of a control.
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