Cells were lysed in chilled radioimmunoprecipitation assay buffer containing 1 mmol/l phenylmethylsulfonyl fluoride (Beijing Dingguo Changsheng Biotechnology Co., Ltd) for 30 min on ice. After centrifugation at 14 000 g/min for 15 min at 4°C, the supernatant was collected and a sample of the supernatant containing 40 µg protein was resolved by SDS-PAGE using 10 % gel. After electrophoresis, the proteins in the gel were transferred to a nitrocellulose membrane (Sangon Biotech Co., Ltd), and the membrane was blocked with 5 % bovine serum albumin fraction V (Roche) in Tris-buffered saline and Tween 20 buffer for 2 h. It was then incubated with the appropriate primary antibody for 3 h at room temperature followed by the corresponding secondary antibody. Signals on the blot were detected by an enhanced chemiluminescence technique (Amersham Life Science).
Phenylmethylsulfonyl fluoride
Manufactured by Dingguo
Sourced in China
Phenylmethylsulfonyl fluoride is a chemical compound commonly used as a protease inhibitor in laboratory settings. It functions by irreversibly inhibiting serine proteases, which are enzymes responsible for cleaving peptide bonds in proteins. This property makes it a valuable tool for preserving protein structure and activity in biological samples during experiments and analysis.
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Lab products found in correlation
2 protocols using phenylmethylsulfonyl fluoride
1
Western Blot Protein Analysis
2
Western Blot Analysis of SP1 Protein
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Total protein was collected from ARPE-19 cells by lysing the cells for 30 min on ice in a cell-lysis buffer [radioimmunoprecipitation assay buffer containing phenylmethylsulfonyl fluoride (Dingguo Changsheng, Beijing, China)], treating the lysates with ultrasound and then centrifuging the lysates at 12,000 rpm for 10 min at 4°C. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Beyotime, Jiangsu, China). Supernatant proteins were collected, denatured, concentrated in 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) stacking gels, separated in 10% SDS-PAGE gels, and transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% skim milk for 1 h, incubated with primary antibodies against SP1 (1 : 150; Santa Cruz Biotechnology, Dallas, TX, USA) and β-actin (1 : 1000; CMC-TAG, Milwaukee, WI, USA) at 4°C overnight, and then incubated with secondary antibodies (1 : 5000; Boster, Wuhan, China) for 40 min. An enhanced chemiluminescence plus kit (Millipore, Billerica, MA, USA) was used to visualize stained bands, and the densities of the grey bands were determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
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