Radioimmunoprecipitation assay (RIPA) lysis buffer (Boster Biotechnology, Wuhan, China) and phenylmethanesulfonyl fluoride (Boster Biotechnology, Wuhan, China) were used to homogenize the hippocampus or cells. The protein concentration was tested by a BCA kit (Beyotime Biotechnology, Haimen, China). Specific monoclonal antibodies anti-EDEM1 (sc-377394), anti-CHOP (sc-7351), anti-Trib3 (sc-365842; 1:1,000 dilution; Santa Cruz, USA), anti-cleaved caspase-3 (9664S), anti-Bcl-2 (3498S), anti-Bax (2772S), and anti-GAPDH (5174S; 1:1,000 dilution; Cell Signaling Technology, USA) were applied to hatched blots overnight at 4°C. Subsequently, the samples were hatched by horseradish peroxidase-labeled second antibody rabbit anti-mouse (5127) or mouse anti-rabbit antibody (93702; 1:5,000 dilution; Cell Signaling Technology, USA) for 2 h. The protein band absorbance was visualized by the Super Signal West Pico Chemiluminescent Substrates (Pierce Biotechnology, Rockford, IL, USA) and quantified by GEL-PRO ANALYZER software (Bio-Rad Laboratories, Hercules, CA, USA).
Phenylmethanesulfonyl fluoride
Manufactured by Boster Bio
Sourced in China
Phenylmethanesulfonyl fluoride is a chemical reagent commonly used in molecular biology and biochemistry laboratories. It functions as a protease inhibitor, effectively preventing the degradation of proteins by inhibiting the activity of serine proteases.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Beyotime (2 mentions)
Ripa lysis buffer, by Boster Bio (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Gel pro analyzer software, by Bio-Rad (2 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti gapdh 5174, by Cell Signaling Technology (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase, by Cell Signaling Technology (1 mentions)
Gsdmd, by Abcam (1 mentions)
3 protocols using phenylmethanesulfonyl fluoride
1
RIPA Lysis Protocol for Protein Analysis
2
Investigating Hippocampal Protein Regulation
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Hippocampal tissue or HCN-2 cells were homogenized using RIPA lysis buffer (Boster Biotechnology, Wuhan, China) containing phenylmethanesulfonyl fluoride (Boster Biotechnology, Wuhan, China) for 15 min. Protein concentrations were then measured with a BCA kit (Beyotime Biotechnology, Haimen, China). Membranes were incubated overnight at 4°C with the following primary monoclonal antibodies: Anti-Nupr1 (ab6028), anti-Cleaved Caspase-3 (ab49822), anti-Bcl-2 (ab196495) (1: 1000 dilution; Abcam, UK), anti-C/EBPβ (2895), anti-IGFBP5 (10941), anti-Bax (2772), and anti-GAPDH (5174) (1:1000 dilution; Cell Signaling Technology, USA). Membranes were then incubated with horseradish peroxidase-labeled secondary mouse anti-rabbit (93702) or rabbit anti-mouse (5127) (1: 5000 dilution; Cell Signaling Technology, USA) antibodies for 2h. Super Signal West Pico Chemiluminescent Substrates (Pierce Biotechnology, Rockford, IL, USA) and GEL-PRO ANALYZER software (Bio-Rad Laboratories, Hercules, CA, USA) were used to visualize and quantify protein band absorbance.
3
Ileum Protein Extraction and Analysis
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Mouse ileum samples were harvested from the small intestine at a distance of 5 cm from the cecum and then homogenated in ice-cold radioimmunoprecipitation assay buffer (Solabio, Beijing, China) supplemented with 1% phenylmethanesulfonyl fluoride (Boster, Wuhan, China). The homogenate was incubated on ice for 30 minutes and then centrifuged at 12,000 g for 10 minutes at 4°C. The tissue protein extract was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. Western blot analysis was performed with GSDMD (Abcam, Cambridge, MA, USA) or glyceraldehyde 3-phosphate dehydrogenase (Cell signaling Technology, Beverly, MA, USA) antibodies.
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