According to the experimental method of Liu et al. (19 (link)), 1 mL of radioimmunoprecipitation assay buffer containing 10 μL of phenylmethylsulfonyl fluoride (Thermo Fisher Scientific, MA, USA) was used to lyse the hepatic and splenic tissue sections for 5 min. They were then spun for 15 min at 12,000 rpm and 4°C. The protein levels in these samples were assessed via bicinchoninic acid assay (Thermo Fisher Scientific), after which they were diluted to 50 μg/mL. The samples were then mixed with sample buffer (4:1) and denatured for 5 min by heating to 100°C. They were then chilled on ice for 5 min. The samples were then separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis using previously prepared gels, with a pre-stained protein ladder used for reference. The proteins were next transferred to polyvinylidene fluoride membranes that were then blocked for 1 h using 5% non-fat milk in TBST. The blots were then incubated with 1 μg/mL primary antibodies of nNOS, eNOS, iNOS, Cu/Zn-SOD, Mn-SOD, CAT, HO-1, Nrf2 and NQO1 at 25°C for 2 h. They were then washed five times with TBST and then incubated with 0.4 μg/mL Goat anti-Mouse IgG secondary antibodies (Thermo Fisher Scientific) at 25°C for 1 h. SuperSignal West Pico PLUS reagent was then used to detect the protein bands with an imaging system (Tanon 5200, Shanghai Tanon Technology Co., Ltd., Shanghai, China).
Phenylmethylsulfonyl fluoride
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
Phenylmethylsulfonyl fluoride is a protease inhibitor commonly used in biochemical research to prevent the degradation of proteins during extraction and purification procedures. It functions by irreversibly inhibiting serine proteases, a class of enzymes that can cleave peptide bonds within proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Cell lysis buffer, by Cell Signaling Technology (3 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (3 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Gradient gel, by Bio-Rad (2 mentions)
Phospho ampk thr172, by Cell Signaling Technology (2 mentions)
Halt s phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Phospho acc ser79, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Cell lysis buffer, by Thermo Fisher Scientific (2 mentions)
37 protocols using phenylmethylsulfonyl fluoride
1
Protein Expression Analysis in Liver and Spleen
2
Western Blot Analysis of HOXA7 Protein
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Whole-cell lysates were prepared using cell lysis buffer (Invitrogen) supplemented with a protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentrations were measured using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific), and then 30 µg of proteins were separated by polyacrylamide gel electrophoresis. After electrotransfer to polyvinylidene difluoride membranes, non-specific binding sites were blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) for 1 hour at room temperature. Membranes were incubated with anti-homeobox A7 (HOXA7) and anti-GAPDH primary antibodies (Abcam, Cambridge, MA, USA) overnight at 4°C, washed with TBST and then incubated with an anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody (Abcam) for 1 hour at room temperature. Immune complexes were visualized using enhanced chemiluminescence. GAPDH was used as an internal control. The gray value of protein bands was analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Exosomal Protein Characterization Protocol
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Exosomal was lysed in Cell lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (Thermo Fisher Scientific, Shanghai, China), a protease inhibitor. Protein concentrations in isolated exosome fractions were measured using a BCA protein assay kit (Thermo Fisher Scientific, Shanghai, China) according to the manufacturer’s instructions. Protein samples were separated with SDS-PAGE, and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA) and blocked with 5% (M/V) skim milk. Primary antibodies were diluted in Antibody Diluent, and membranes were incubated with an antibody overnight at 4°C. antibodies specific for the designated antigens and purchased from Abcam, ShangHai, China: CD63 (1:1000, ab134045), CD9 (1:1000, ab263019), TSG101 (1:2000, ab125011). Next, After washing 3 times in TBST, Anti-rabbit IgG, HRP-linked Antibody (1:2000, Bioss, Beijing, China:) was added for 1 hour at room temperature, and blots were developed with ECL detection reagents (Thermo Fisher Scientific, Shanghai, China). Band intensities on exposed flms were quantifed using Image J software (NIH, USA).
4
Western Blot Analysis of PCNA and p21
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The protein precipitates were extracted using an RIPA protein lysate (cat no. 89900, Thermo Fisher) containing a protease inhibitor phenylmethylsulfonyl fluoride (cat no. 36978, Thermo Fisher, USA). Protein concentrations were determined using a BCA kit (ThermoFisher, USA). Then, the protein was subjected to SDS polyacrylamide electrophoresis and transferred onto a PVDF membrane (cat no. IPVH00010, Millipore). The PVDF membrane was blocked with 5% skimmed milk at room temperature. Then, the PVDF membranes were incubated overnight at 4 ∘C with primary antibodies diluted with 5% skimmed milk. The used primary antibodies were mouse anti-PCNA monoclonal antibody (1:2000, cat no. ab29, Abcam, USA) and rabbit anti-p21 polyclonal antibody (1:1000, cat no. 10355-1-AP, Proteintech, Chian). Rabbit anti-vinculin monoclonal antibody (1:5000, cat no. ab129002, Abcam, USA) was used as the loading control. Then, the appropriate secondary antibody (CoWin Biosciences) was incubated with the PVDF membranes based on the genetic origin of the primary antibody. A gel imaging system was used to scan the protein bands using ECL reagents, and vinculin was used as an internal reference.
5
Macrophage Polarization Regulation
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Dulbecco’s Modified Eagle’s Medium, Minimum Essential Media, and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). Zinc protoporphyrin IX (ZnPP) and a primary antibody against HO-1 were the products of Enzo Life Sciences (Farmingdale, NY, USA). Primary antibodies for detecting Nrf2 (ab137550) and CD11b (ab8878) were obtained from Abcam (Cambridge, MA, USA). Anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibodies were provided by Thermo Fisher Scientific (Eugene, OR, USA). Antibodies for TruStain FcX (anti-mouse CD16/32), CD45, CD11b, F4/80, Ly6C, Ly6G, CD86, CD36, and CD206 for flow cytometric analysis were purchased from Biolegend (San Diego, CA, USA). 4’,6-Diamidino-2-phenylindole and phenylmethylsulfonyl fluoride were purchased from Thermo Fisher Scientific and Sigma-Aldrich (St. Louis, MO, USA), respectively.
6
Enrichment and proteomic analysis of spermatocyte proteins
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The pachytene and diplotene spermatocytes were enriched using STA-PUT method [25 (link)] and then were lysed in cold RIPA buffer (Beyotime, P0013C) with phenylmethylsulfonyl fluoride (Thermo Fisher, 36978) and protease inhibitor cocktail (Roche, 04693116001). The lysates were sonicated for 8–10 cycles (3 s on/off) with 8% pulses and centrifuged at 15,000 xg in 4°C for 15 minutes. The supernatant was divided into two aliquots, and each aliquot was incubated with pre-cleared Protein A/G agarose beads (Santa Cruz, sc-2003) and 2 μg anti-MTL5 C-terminal antibody (epitope from residues 221–475) or rabbit IgG non-specific antibody (ABclonal, AC005). After overnight incubation at 4°C, the agarose beads were washed in RIPA buffer for 3 times and twice in 10 mM Tris buffer, and then the immunocomplexes were dissociated from the beads with elution buffer (0.2 M Glysine, 0.15% NP-40, pH 2.3) at room temperature. After validation of the eluted samples by silver staining, samples were analyzed by mass spectrometry in National Center for Protein Science Shanghai. The candidate interacting proteins of MTL5 are listed in S1 Table .
7
Analyzing Cardiac Protein Expression
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Mouse heart tissues were homogenized and lysed with radioimmunoprecipitation assay lysis buffer (KeyGEN BioTECH, Nanjing, China) supplemented with 1% phenylmethylsulfonyl fluoride (36978; ThermoFisher) and Pierce protease and phosphatase inhibitor (88668; ThermoFisher). Denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene fluoride membranes. After being blocked with 5% non-fat milk, the membranes were incubated overnight at 4 °C with primary antibodies against LYVE-1 (ab14917; Abcam), Podoplanin (ab11936; Abcam), VEGFR3 (A10971; ABclonal, Wuhan, China), and IGF-1 (28530-1-AP; Proteintech, Wuhan, China) from 1:500 to 1:1000 dilution. The membranes were then incubated with HRP-conjugated secondary antibodies, and protein blots were developed with an enhanced chemiluminescence kit. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (AP0063; Bioworld, Nanjing, China; 1:1000 dilution) and β-actin (AC026; Abclonal; 1:1000 dilution) were used as loading controls. Finally, protein band densities were quantified using ImageJ software (NIH).
8
Quantification of aortic tissue proteins
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Aortic tissues and 20 μM montelukast-induced macrophages were homogenized using an ultrasonic disintegrator (Sonics & Materials, Inc., Newtown, CT, USA) in protein extraction buffer (CytoBuster; Novagen, Merck KGaA, Darmstadt, Germany) with 20 mM of ethylenediaminetetraacetic acid (Dojindo, Kumamoto, Japan) and 1 mM of phenylmethylsulfonyl fluoride (Thermo Fisher Scientific). Lysate protein concentration was measured using the Qubit® Protein Assay Kit and Qubit® 2.0 fluorometer (Thermo Fisher Scientific). An equal concentration of total protein was applied to each assay kit and detected according to the manufacturer's protocol for each ELISA kit (TIMP-1 and TGF-β1: R&D Systems, Minneapolis, MN, USA; TIMP-2: RayBiotech, Norcross, GA, USA; IGF-1: Mediagnost, Reutlingen, Germany; IL-1β, IL-6, TNF-α, and MCP-1: Bender MedSystems, Vienna, Austria; IL-10: Thermo Fisher, Waltham, MA, USA) [12 (link)]. Seven samples were analyzed per group.
9
Isolation of Brush Border Membrane Vesicles
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The proximal half of the small intestine was removed and flushed with cold phosphate-buffered saline supplemented with 0.1 mM phenylmethylsulfonyl fluoride (Sigma) to arrest GLUT2 in the apical membrane. Mucosa was scraped and homogenized with a polytron in 15 mL of homogenization buffer (300 mM mannitol, 5 mM EGTA, 12 mM Tris-HCl, pH 7.1) supplemented with Halt protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA) and 1 mM phenylmethylsulfonyl fluoride. Next, 20 mL of ice-cold water with 20 mM MgCl2 was added to each homogenate and the resultant samples were mixed by inversion (15 minutes at 4°C) for divalent cation precipitation. Samples were then centrifuged at 3000g (15 minutes at 4°C) to separate aggregated membranes, supernatants transferred to new tubes and centrifuged at 30 000g (30 minutes at 4°C). Pellets were resuspended in 35 mL of buffer containing 150 mM mannitol, 2.5 mM EGTA, 6 mM Tris-HCl, pH 7.1. MgCl2 was added to a final concentration of 20 mM, and samples were mixed and centrifuged exactly as described above. Pellets containing brush border membrane vesicles (BBMVs) were resuspended in 50 µL of sterile phosphate-buffered saline and used for analyses.
10
HeLa Cell Transfection Protocol
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HeLa cells (RIKEN Cell Bank, Ibaraki, Japan) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37 °C under 5% CO2. Cells were passaged regularly to maintain exponential growth. Lipofectamine RNAiMAX (2 μL; Invitrogen) was added to a mixture of 1 μg RNA and 145 μL Opti-MEM I solution (Invitrogen) in a well of a 12-well plate. After incubation at room temperature for 15 min, 8.5 × 104 HeLa cells suspended in DMEM containing 10% FBS (0.85 mL) were added to the RNA solution. After incubation for 24 h at 37 °C, the cells were collected and lysed with 0.2 mL of 1× Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA) containing 100 mM phenylmethylsulfonyl fluoride (Thermo Fisher Scientific). The lysates (7.5 μL) were subjected to SDS-PAGE (10–20% gradient gel, Atto) and western blot analysis as described above.
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