0.2 μm polyvinylidene difluoridemembrane

Manufactured by Bio-Rad

The 0.2 μm polyvinylidene difluoride (PVDF) membrane is a microporous membrane made of PVDF material. The membrane has a pore size of 0.2 μm, which is suitable for various filtration and separation applications.

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3 protocols using 0.2 μm polyvinylidene difluoridemembrane

Biotin-labeled Protein Detection by Western Blotting

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Samples were separated by SDS-PAGE as above. Protein
bands were transferred to a 0.2 μm polyvinylidene difluoride
membrane (Bio-Rad) by electroblotting at a constant current of 1.3
A for 7 min on Trans-Blot Turbo (Bio-Rad). After transfer, biotin-labeled
proteins were detected using Cy3–streptavidin conjugate. Blots
were blocked against nonspecific reactions by soaking in Bullet Blocking
One for Western Blotting (Nacalai Tesque, Inc.) (50 mL/gel) at room
temperature for 30 min. The blots were incubated with Cy3–streptavidin
(Jackson ImmunoResearch Laboratories, Inc.) (1.0 mg/mL, 10 μL/gel)
in TBS-T buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.6)
(50 mL) at room temperature for 1 h on a shaker. The blots were then
washed with TBS-T (50 mL) at room temperature for 5 min. This washing
procedure was repeated three times. Fluorescent gel images were acquired
on a Typhoon 9400 scanner (Amersham Biosciences/GE Healthcare) using
a 532 nm laser and an emission filter of 570 nm BP20.

Matsushita T., Maruyama N., Koyama T., Hatano K, & Matsuoka K. (2022). Modification of Fab Fragments by Dibromopyridazinediones Carrying Mono- and Double-Biotin Functionalities. ACS Omega, 7(38), 34554-34562.

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Comparative Analysis of Cx36 and Cx43 Protein Levels

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Tissue samples were sonicated in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% Na deoxycholate, 50 mM Tris), and total protein was determined using BCA protein assays (Thermo-Scientific, Waltham, MA). Experiments were performed on the XCell II Blot Module (Invitrogen). From each sample, 20 μg of protein was loaded into a lane on a NuPage 4–12% Bis-Tris gel (Life Technologies, Carlsbad, CA), transferred to 0.2-μm polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), and processed with a blocking solution of 3% BSA and 5% nonfat dry milk in 1 × tris-buffered saline and Tween-20 (TBST). Rabbit anti-Cx36 (0.5 μl/ml; Invitrogen), rabbit anti-Cx43 (0.2 μl/ml; Invitrogen), and mouse anti-tubulin (1:10,000; Sigma-Aldrich, St. Louis, MO) were used as primary antibodies, and were visualized with horseradish peroxidase-conjugated anti-rabbit antibody (1:10,000; Invitrogen) or anti-mouse IgG antibody (1:10,000; Sigma-Aldrich). Signals were enhanced using enhanced chemiluminescence detection reagents (Thermo-Scientific) and detected on a BioSpectrum imaging system (UVP, Upland, CA). Densities were quantified using VisionWorks (UVP) and normalized relative to tubulin.

Wu J., Gao M., Rice S.G., Tsang C., Beggs J., Turner D., Li G., Yang B., Xia K., Gao F., Qiu S., Liu Q, & Kerrigan J.F. (2016). Gap Junctions Contribute to Ictal/Interictal Genesis in Human Hypothalamic Hamartomas. EBioMedicine, 8, 96-102.

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Tendon Cell Protein Isolation and Analysis

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Tendon-derived cells were treated with 1 mM sodium orthovanadate (Sigma-Aldrich) for phosphatase inhibition and then scraped in a cell lysis buffer that contains 0.5 mM sodium orthovanadate, 32 mM Tris base (Fisher Scientific), 50 mM (5%) glycerol (Sigma-Aldrich), 34.7 mM (1%) sodium dodecyl sulphate (SDS; Fisher Scientific) and 0.5% Protease Inhibitor Cocktail Set III (Merck Millipore). Protein separation was performed by electrophoresis by loading approximately 2.5 μg of protein per well to 10% polyacrylamide gel electrophoresis gels (BioRad). The amounts of the proteins loaded were measured by a bicinchoninic acid assay. Proteins were transferred from gels on to a 0.2 μm polyvinylidene difluoride membrane (BioRad) using a Western blot transfer system (BioRad). Molecular weight was indicated by Precision Plus Protein Dual Color Standards (BioRad). The details of the antibodies used and their working dilutions are shown in Supplementary Table 2. GAPDH was used as the endogenous housekeeper. Anti-rabbit IgG antibody was used as the secondary antibody. Proteins were detected by enhanced chemiluminescence (GE Healthcare Life Sciences) and visualised using Alliance 6.7 system and NineAlliance 4.7 17.00 (uvitec).

Morita W., Snelling S.J., Wheway K., Watkins B., Appleton L., Carr A.J, & Dakin S.G. (2019). ERK1/2 drives IL-1β-induced expression of TGF-β1 and BMP-2 in torn tendons. Scientific Reports, 9, 19005.

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