Mouse tissues or cultured cells were lysed using lysis buffer (125 mmol/l NaCl, 20 mmol/l Tris–HCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate; pH 7.4) followed by centrifugation at 15,000 rpm for 5 min at 4 °C to remove undissolved debris. The lysates were then sonicated (Handy Sonic UR-20 P; Tomy Seiko Co, Ltd) on ice. Protein concentrations of the lysates were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were loaded onto SDS–PAGE gels, transferred to polyvinylidene difluoride membranes (Wako), and incubated overnight with the primary antibody of interest. Alkaline phosphatase–conjugated secondary antibody (Cell Signaling) and 5-bromo-4-chloro-3-indolyl-phosphate–nitro blue tetrazolium (Wako) were used to detect the target proteins. The primary antibodies used for immunoblotting are listed in Table S2 . Coomassie brilliant blue staining was conducted using Rapid CBB KANTO (KANTO Chemical Co) according to the manufacturer’s instructions.
Polyvinylidene difluoride membranes
Manufactured by Fujifilm
Polyvinylidene difluoride (PVDF) membranes are a type of laboratory equipment used for various applications. They are made of a chemically resistant and durable polymer material. PVDF membranes are commonly used for filtration, separation, and purification processes in research and diagnostic settings.
Automatically generated - may contain errors
Lab products found in correlation
Rapid cbb kanto, by Kanto Chemical (1 mentions)
Alkaline phosphatase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Handy sonic ur 20p, by TOMY (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
2 protocols using polyvinylidene difluoride membranes
1
Western Blot Analysis of Mouse Proteins
2
Butyrate and Propionate Effects on Ca9-22 Cells
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Ca9-22 cells (2 × 10 5 cells/well) were treated for 48 h with 400 µL of S7-supplemented Epi-Life medium containing several concentrations of butyrate or propionate in the presence or absence of N-acetylcysteine or ascorbic acid. Supernatants (20 µL) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins in the gels were transferred to polyvinylidene difluoride membranes (Wako). After blocking with EzBlockChemi (Atto, Tokyo, Japan), membranes were treated with a series of primary and secondary antibodies labeled with horseradish peroxidase. The band images were developed with Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA) and images were photographed using ChemiDoc XRS (Bio-Rad).
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