Western blotting was performed on 7 SCSTs, 9 benign and 7 malignant OGCTs. Total protein lysates were prepared using NP-40 lysis buffer (Thermo Fisher Scientific) with addition of 1 mM phenylmethanesulfonyl (Sigma-Aldrich, St. Louis, MQ, USA) and protease inhibitor (complete protease inhibitor cocktail; Sigma-Aldrich). The concentrations were measured using the Pierce BCA Protein assay kit (Thermo Fisher Scientific). Protein lysates of 30 μg were separated in NuPAGE Novex 4–12% Bis-Tris gel and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk diluted in Tris-buffered saline/0.05% Tween-20 prior to incubation with anti-BECN1 rabbit polyclonal (Novus Biologicals; dilution 1:1,000) or anti-GAPDH rabbit monoclonal (Cell Signaling Technology; dilution at 1:1,000) antibodies overnight at 4°C. Anti-rabbit IgG-HRP (Cell Signaling Technology; dilution at 1:2,000) was used as a secondary antibody. Signals were detected using the Novex ECL HRP chemiluminescent substrate reagent (Thermo Fisher Scientific) and LI-COR Odyssey Fc Imaging system (LI-COR Biosciences, Lincoln, NE, USA) and quantified using Image Studio Lite version 5.2 (LI-COR Biosciences).
Phenylmethanesulfonyl
Manufactured by Merck Group
Sourced in United States
Phenylmethanesulfonyl is a chemical compound used as a reagent in organic synthesis. It is a colorless, crystalline solid with a melting point of approximately 92-94°C. The compound is primarily used as a protecting group for amino groups in various chemical reactions.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
A2066, by Merck Group (2 mentions)
Supersignalwest femto maximum sensitivity substrate system, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Np40 lysis buffer, by Thermo Fisher Scientific (1 mentions)
Odyssey fc imaging system, by LI COR (1 mentions)
Image studio lite version 5, by LI COR (1 mentions)
Rabbit monoclonal anti gapdh, by Cell Signaling Technology (1 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
Ab3594, by Cell Signaling Technology (1 mentions)
Ab1791, by Abcam (1 mentions)
Protease inhibitor mixture, by Merck Group (1 mentions)
Ip lysis wash buffer, by Thermo Fisher Scientific (1 mentions)
Microson, by Bioventus (1 mentions)
Na3vo4, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
3 protocols using phenylmethanesulfonyl
1
Western Blot Analysis of BECN1 in Ovarian Tumors
2
Quantitative Immunoblotting Analysis
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Cells were homogenized in IP Lysis/Wash buffer (Thermo Fisher) supplemented with 5% (vol/vol) Protease Mixture Inhibitor (Sigma) and 1 mM phenylmethanesulfonyl (Sigma). After two homogenization cycles (7 s) with an ultrasonic cell disruptor (Microson; Misonix), total cell lysates were centrifuged 10 min at 18,000g, and supernatant containing proteins was collected. The protein concentration of the extracts was determined by Pierce BCA Protein Assay Kit (Thermo Scientific). Immunoblotting analyses were performed as described in previous studies3 (link). Antibodies against Actin (Sigma, A2066; dilution 1:4,000), active b-catenin (Merck Millipore, 05-665, clone 8E7; dilution 1:1,000), total β-catenin (BD biosciences, 610154, clone 14/β-catenin; dilution 1:2,000), DOT1L (Bethyl, A300-954A; dilution 1:1,000), GAPDH (Ambion, AM4300, clone 6C5; dilution 1:10,000), total H3 (Abcam, ab1791; dilution 1:10,000), H3K79me2 (Abcam, ab3594; dilution 1:1,000), and SIRT1 (Cell signalling, 2496, clone C14H4; dilution 1:1,000) were used following manufacturer’s instructions. The blotting signals were detected using the SuperSignalWest Femto Maximum Sensitivity Substrate system (Thermo Scientific).
3
Protein Extraction and Western Blot Analysis
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Cells were harvested and resuspended in RIPA buffer (Thermo Scientific) supplemented with 1 mM phenylmethanesulfonyl (Sigma), 5% protease inhibitor cocktail (Sigma), 2.3 mM Na 3 VO 4 (Sigma) and 5 mM NaF (Merck Millipore). Cell lysates were sonicated (two cycles of 7 s) and centrifuged at 18.000 g for 10 min, and supernatant containing proteins was collected. Protein concentration of the extracts was determined by Pierce BCA Protein Assay Kit (Thermo Scientific). Immunoblotting analyses were performed as previously described 19 . Antibodies against Actin (Sigma, A2066; dilution 1:4.000), active b-catenin (Merck Millipore, 05e665, clone 8E7; dilution 1:1.000) and total b-catenin (BD biosciences, 610154, clone 14; dilution 1:2.000) were used. Blotting signals were detected using the SuperSignalWest Femto Maximum Sensitivity Substrate system (Thermo Scientific).
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