Total protein of cell was extracted and protein concentration was measured using the BCA (bicinchoninic acid) protein quantitation kit (Thermo Fisher Scientific, MA, USA). A total of 20μg protein from each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto 0.45 μm polyvinylidene difluoride membranes (Bio-Rad Laboratories, CA, USA). Then, the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies for Notch1 (D1E11; #3608s; 1:1000 dilution), E-cadherin (4A2; #14472s; 1:1000 dilution), Vimentin (D21H3; #5741s; 1:1000 dilution), Cleaved Notch1 (Val1744; D3B8; #4147; 1:1000 dilution), c-Myc (D3N8F; #13987; 1:1000 dilution) and β-actin (8H10D10; #3700s; 1:1000 dilution) were purchased from Cell Signaling Technology (Danvers, MA, USA). The membranes were incubated with secondary horseradish peroxidase (HRP)-conjugated goat anti-mouse (ab6789, 1:10,000 dilution, ABCAm, Cambridge, UK) and goat anti-rabbit antibodies (ab6721, 1:10,000 dilution, ABCAm, Cambridge, UK) at room temperature for 2 hrs. The bands were detected using an enhanced chemiluminescence reagent and visualized with a Fusion FX7 System (Vilber Lourmat, France). ImageJ software was used to calculate the intensity (gray value) of each protein band, which was normalized to that for GAPDH.
0.45 μm polyvinylidene difluoride membranes
Manufactured by Bio-Rad
Sourced in United States
The 0.45 μm polyvinylidene difluoride (PVDF) membranes are a type of lab equipment used for filtration and separation purposes. These membranes have a pore size of 0.45 micrometers and are made of the polymer polyvinylidene difluoride.
Automatically generated - may contain errors
Lab products found in correlation
Protein quantitation kit, by Thermo Fisher Scientific (1 mentions)
Ab6789, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
C myc d3n8f, by Cell Signaling Technology (1 mentions)
Fusion fx7 system, by Vilber (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence system, by GE Healthcare (1 mentions)
β tubulin, by Merck Group (1 mentions)
Non fat dry milk, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Caveolin 1, by BD (1 mentions)
Chemidoc xrs image analyzer, by Bio-Rad (1 mentions)
Western blocker solution, by Merck Group (1 mentions)
West pico plus hrp substrate, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
M per, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti rabbit igg ab, by Abcam (1 mentions)
Rabbit anti gapdh ab, by Abcam (1 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
6 protocols using 0.45 μm polyvinylidene difluoride membranes
1
Immunoblotting Analysis of Notch Signaling
2
Western Blot Analysis of Vascular Proteins
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After treatment, the PASMCs were lysed using RIPA buffer (Pierce) supplemented by 5% protease inhibitor cocktail (Sigma-Aldrich). The total protein concentration was determined by BCA kit (Pierce). Homogenates were denatured by adding 150 mM dithiothreitol (DTT) and heating at 95°C for 3 min. Whole cell lysates were separated on SDS-PAGE calibrated with Precision Plus protein dual color molecular weight markers (Bio-Rad). The proteins were then transferred onto 0.45 μm polyvinylidene difluoride membranes (Bio-Rad), blocked with 5% nonfat dry milk (Bio-Rad) in Tris-buffered saline containing 0.2% Tween 20 (TBST), and blotted with primary antibodies against Caveolin-1 (BD Biosciences), p21 (BD Biosciences), LC3B (Cell Signaling Technology), or beta-tubulin (Sigma-Aldrich). The membranes were then washed by TBST×3 and incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Kirkegaard and Perry Laboratories). The bands were detected using an enhanced chemiluminescence system (GE healthcare). Gray density of each image was analyzed by Image J; data were calculated as a ratio of the protein of interest to house-keeping protein beta-tubulin, serving as internal control.
3
Western Blot Analysis of eIF2α
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Samples and controls were loaded and separated with stain-free 4 to 20% gels (Bio-Rad). Proteins were transferred onto 0.45-μm polyvinylidene difluoride membranes (Bio-Rad) using the Trans-blot turbo transfer system (Bio-Rad). The blot was blocked using Western blocker solution (Sigma-Aldrich) and incubated in either anti-total eIF2α (1:1,000), anti-puromycin (1:1,000), or eIF2α-phosphorylated (1:1,000; Ser-51; Invitrogen), followed by incubation with either anti-mouse (1:5,000) or anti-rabbit (1:5,000). Both secondary antibodies were horseradish peroxidase (HRP) conjugated, visualized using West Pico plus HRP substrate (Thermo Fischer), and measured with a ChemidoC XRS image analyzer (Bio-Rad).
4
Protein Extraction and Western Blot Analysis of UPR Markers
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Following treatment with recombinant TNF-α for 24 hours in serum-free medium, protein was extracted from SaOs-2 cells at 4°C using a mammalian protein extraction reagent (M-PER; Cat No.78501; Thermo Fisher Scientific) containing protein inhibitors. The protein concentration was estimated according to the Bradford’s method using BSA as a standard.28 (link) Equal amounts of protein were heated to 95°C for five minutes and subsequently separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands were electroblotted onto 0.45 μm polyvinylidene difluoride membranes (Bio-Rad, Hercules, USA). The membranes were blocked with 5% BSA for one hour and subsequently incubated with anti-IRE-1α, anti-Bip, anti-Ero1-Lα, and anti-ß-actin antibodies overnight at 4°C. The membranes were subsequently incubated with horseradish peroxidase conjugated secondary antibodies. The immunoreactive bands were visualized using an enhanced chemiluminescence kit (Pico PLUS, Cat No.34580; Thermo Fisher Scientific) .
5
Western Blot Analysis of Hepcidin and OmpA
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Hepcidin, OmpA as well as the reference protein (GAPDH) sample was separated by 12% SDS-PAGE and transferred onto 0.45-μm polyvinylidene difluoride membranes (Bio-Rad Laboratories) for 90 min at 350 mA, and then blocked with TBST buffer (50 mM Tris-HCl, 150 mM NaCl and 0.1% Tween-20, pH 7.4) containing 5% non-fat dry milk (w/v) at 4 °C overnight. After washing with TBST three times for 30 min, the blots were incubated with rabbit anti-hepcidin Ab (1:1,000), rabbit anti-OmpA Ab (1:1,000) or rabbit anti-GAPDH Ab (Abcam. 1:3,000). The blots were washed thrice with TBST for 30 min, and then incubated for 1 h with the HRP-conjugated goat anti-rabbit IgG Ab (Abcam) at room temperature. After washing with TBST, the immunoreactive proteins were visualized using a chemical luminescent immunodetection system (Tanon 4500, Tanon Technology Company).
6
Western Blot Analysis of Proteins
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Proteins isolated from tissues or cells were lysed in radioimmunoprecipitation buffer containing protease inhibitors (Roche Diagnostics) and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA). Proteins isolated from tissues (30 μg) or cells (4–6 μg) were separated by 5%–20% sodium dodecyl sulfate (SDS)-PAGE (DRC, Tokyo, Japan) using Precision Plus Protein™ Dual Color standards (Bio-Rad, Hercules, CA), and transferred to 0.45 μm polyvinylidene difluoride membranes (Bio-Rad). After blocking with 5% skim milk, the membranes were incubated with primary antibodies overnight at 4°C (Supplementary Table S2 ). The membranes were then incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies (1:4000; Dako; Agilent Technologies, Santa Clara, CA). Luminescence was quantified on an LAS-4000 luminescent image analyzer (Fujifilm Corp., Tokyo, Japan) coupled to image analysis software (Multi Gauge; Fujifilm Corp.). The staining intensity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.
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