NRCMs or cardiac tissues were lysed in lysis buffer [50 mM tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 1% SDS, and 0.5% sodium deoxycholate] plus phenylmethylsulfonyl fluoride (PMSF) (Solarbio) on ice for 20 min. Then, the samples were centrifuged at 12,000 rpm at 4°C for 15 min to obtain the protein extract. After the protein concentration was quantitated by bicinchoninic acid (Thermo Fisher Scientific), 2 μg of appropriate primary antibody and protein A–Sepharose (Amersham Biosciences) were added to the protein samples with gentle shaking at 4°C for 24 hours, followed by centrifugation at 5000 rpm at 4°C for 10 min. The pellet was washed twice with wash buffer I [50 mM tris-HCl (pH 7.5), 500 mM sodium chloride, 0.1% NP-40, and 0.05% sodium deoxycholate] and once with wash buffer II [50 mM tris-HCl (pH 7.5), 0.1% NP-40, and 0.05% sodium deoxycholate]. Bound proteins were eluted by boiling beads in 2× sample buffer, and the precipitated proteins were subjected to SDS-PAGE. Immunoblot data were quantified and analyzed with a Gel-Pro 4.5 analyzer (Media Cybernetics, USA).
Phenylmethylsulfonyl fluoride pmsf
Manufactured by Solarbio
Sourced in China
Phenylmethylsulfonyl fluoride (PMSF) is a protease inhibitor commonly used in biochemical research. It functions by irreversibly inhibiting serine proteases, which are enzymes that can break down proteins. PMSF is often used to prevent protein degradation during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Gel pro 4.5 analyzer, by Media Cybernetics (1 mentions)
Protein a sepharose, by Cytiva (1 mentions)
Gsdmd n, by Affinity Biosciences (1 mentions)
Tunel detection kit, by Beyotime (1 mentions)
Cleaved caspase 1, by ABclonal (1 mentions)
Ml385, by MedChemExpress (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Ros detection kit, by Beyotime (1 mentions)
β actin, by ABclonal (1 mentions)
Il 1β, by ABclonal (1 mentions)
Phosphatase inhibitor, by Applygen (1 mentions)
Skim milk powder, by Beyotime (1 mentions)
Stop solution, by Abcam (1 mentions)
Sds page sample loading buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
7 protocols using phenylmethylsulfonyl fluoride pmsf
1
Immunoprecipitation of Cardiac Proteins
2
Airborne Particulate Matter Analysis
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PM (The standard reference airborne 1649b) was purchased from the National Institute of Standards and Technology (NIST, Gaithersburg, USA). PAL (powder) was obtained from Macklin (Shanghai, China). TUNEL detection kit and ROS detection kit were purchased from Beyotime (Shanghai, China). The ELISA kits of IL-18, IL-6, IL-8, SOD, MDA, and IL-1β were purchased from Boyun Biotechnology (Shanghai, China). The DMEM/F12 medium, PBS, and penicillin-streptomycin were purchased from Gibco (USA), and the fetal bovine serum (FBS) was purchased from Sijiqing (Hangzhou, China). RIPA lysis buffer and phenylmethylsulfonyl fluoride (PMSF) were purchased from Solarbio (Beijing, China) while the phosphatase inhibitors were purchased from Applygen (Beijing, China). Antibodies against Nrf2, ASC, IL-18, IL-1β, Cleaved-caspase-1, and β-actin were purchased from Abclonal (Wuhan, China); antibodies against NLRP3, HO-1, NQO1, and N-GSDMD were purchased from Affinity (Jiangsu, China); antibody against EpCAM was purchased from Santa (Cruz, USA); the secondary antibody for Western Blotting was purchased from Biosharp (Beijing, China); ML385 was purchased from MedChemExpress (Shanghai, China).
3
Detailed Antibody Analysis Protocol
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Detailed information on antibodies was listed in Supplementary Table S4 . Phosphate buffered saline (PBS), Tween‐20, RIPA lysis buffer, phenylmethylsulfonyl fluoride (PMSF), phosphotungstic acid negative stain solution (2%), and glutaraldehyde (2.5%, EM Grade) were obtained from Solarbio (Beijing, China). Methanol was obtained from Concord Technology (Tianjin, China). Sodium dodecyl sulfate (SDS), BCA Protein Assay Kit, SDS‐PAGE sample loading buffer, prestained colour protein marker, nitrocellulose membrane, polyvinylidene difluoride membrane, skim milk powder, and BeyoECL Plus were obtained from Beyotime Biotechnology (Shanghai, China). Precast Protein Improve Gels (4%–20%, 10 wells) were obtained from Yeasen (Shanghai, China). The coating buffer was obtained from BioLegend (California, USA). Casein blocking buffer (1%) was obtained from Leagene Biotechnology (Beijing, China). Tris(hydroxymethyl) aminoethane, 2‐[4‐(2‐hydroxyethyl)‐1‐piperazinyl]ethanesulfonic acid (HEPES), ethylene diamine tetraacetic acid (EDTA), and glycine was obtained from Aladdin (Shanghai, China). TMB chemiluminescence chromogenic solution and stop solution were obtained from Abcam (Cambridge, MA).
4
Apoptosis Detection in Cell Cultures
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CoCl2, 2,7-dichlorodihyfluorescein diacetate (DCFH-DA), Tris, glycine, Tween-20, SDS, acridine orange (AO), propidium iodide (PI), anti-microtubule associated proteins 1A/1B light chain 3B (LC3I/II; cat. no. L7543) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Trypsin-EDTA was obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Trypsin solution without EDTA was purchased from Beyotime Institute of Biotechnology (Hangzhou, China). The Annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit was purchased from BD Biosciences (San Jose, CA, USA). Primary antibodies were purchased from CST Biological Reagents, Co., Ltd. (Shanghai, China) or Abcam (Cambridge, UK). Secondary antibodies were purchased from OriGene Technologies, Inc. (Beijing, China). All other chemicals were obtained from Sigma-Aldrich (Merck KGaA). Radioimmunoprecipitation assay (RIPA) lysis buffer and phenylmethylsulfonyl fluoride (PMSF) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).
5
Western Blot Analysis of Testicular Proteins
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The testicular tissues were lysed in RIPA buffer (150 mM NaCl; 50 mM Tris-Cl, pH 8; 1% NP-40; 0.5% deoxycholate; 0.1% SDS) (Solarbio, Beijing, China) with phenylmethylsulfonylfluoride (PMSF, final concentration 1 mM) (Solarbio, Beijing, China). The resulting samples were separated using 10% SDS-PAGE and transferred to PVDF membranes. After blocking for 1 h in 5% non-fat milk in TBST at 25 °C, the membranes were incubated with specific primary antibodies overnight at 4 °C. The primary antibodies used were KDM1A (Novus, Centennial, CO, USA, 1:1000), KDM4B (Abcam, Cambridge, UK,1:2000), β-tubulin (Zen Bioscience, Chengdu, China, 1:5000). The primary antibody binding was visualized with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:10,000, ZSGB-BIO, Beijing, China) for 1 h at 25 °C. The signal intensities were measured using Chemiluminescent HRP Substrate (Millipore, Billerica, Massachusetts, USA) and image analysis software (ImageJ1.8.0, NIH, Bethesda, MD, USA).
6
Immunomodulatory Mechanisms in Cell Culture
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Dulbecco's modified eagle's medium (DMEM), fetal bovine serum (FBS), 0.25% trypsin, 0.03% ethylenediaminetetraacetic acid solution, and Ham F-12 were purchased from Gibco (San Diego, CA, USA). RNAiso Plus, reverse transcription-polymerase chain reaction (RT-PCR) kits and SYBR Premix Ex Taq™ (Tli RNaseH Plus) were purchased from TaKaRa (Dalian, China). Monoclonal mouse anti-human IDO antibody (Millipore, Billerica, MA) and polyclonal rabbit anti-human Dectin-1 were obtained from Cell Signaling Technology (Boston, MA, USA). 1-methyl tryptophan (1-MT), curdlan and laminarin were obtained from Sigma-Aldrich. Bicinchoninic acid assay and ECL western blotting detection reagents were purchased from Beyotime (Shanghai, China). Phenylmethylsulfonyl fluoride (PMSF) and cell lysis buffer (RIPA) were purchased from Solarbio (Beijing, China).
7
Synthesis and Characterization of Zinc-based Nanomaterials
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Zinc acetate and 2-methylimidazole (2-MeIM) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Glucose oxidase (GOx), ethanol, and 4,4-azodianiline were purchased from Aladdin Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO), and mesitylene (trimethyl benzene, TMB) were purchased from Sigma–Aldrich (Shanghai, China) Trading Co., Ltd. Phosphate-buffered saline (PBS, 10×), and Dulbecco's modified Eagle's medium (DMEM) were purchased from KeyGEN (Nanjing, China). Fetal bovine serum (FBS) was purchased from ExCell Bio (Taicang, China). Penicillin/streptomycin (P/S, 100 ×), Bovine albumin (BSA), and DAPI stain solution were purchased from Sangon Biotech (Shanghai, China) Co., Ltd. Cell Counting Kit-8 (CCK-8) was purchased from TargetMol (Boston, MA, USA). JC-1 kit, propidium iodide (PI), and calcein-AM dyes, Annexin V-FITC/PI Apoptosis Detection Kit (Cat No. 40302ES50) were acquired from Yeasen Biotechnology (Shanghai, China). 2 × Phanta Max Master Mix, ChamQ SYBR® qPCR Master Mix (High ROX Premixed), and FastPure Cell/Tissue DNA Isolation Mini Kit were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). 0.05% trypsin solution was purchased from YIFEIXUE BIOTECH (Nanjing, China). RIPA buffer and Phenylmethylsulfonyl fluoride (PMSF) were purchased from Solarbio (Beijing, China).
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