All cells were lysed with radioimmunoprecipitation assay (RIPA) buffer: 20 mM Tris pH 7.4, 150 mM NaCl, 0.5% deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM PMSF, and a mixture of protease inhibitors (Complete, EDTA-free, Roche Applied Science). Lysates were cleared of insoluble material by centrifugation. As noted, lysates were immunoprecipitated with the indicated antibodies using protein G sepharose beads (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Samples were electrophoresed through polyacrylamide-SDS gels and then transferred to Immuno-Blot polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked in phosphate-buffered saline containing 0.05% Tween and 5% bovine serum albumin and then incubated with antibodies against the target proteins. After washing, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibodies (Jackson Immuno Research, West Grove, PA, USA) before detection with a chemiluminescent substrate. Full images for all cropped blots are provided in Additional file 1 and Additional file 2 .
Immunoblot polyvinylidene fluoride pvdf membranes
Manufactured by Bio-Rad
Sourced in United States
Immunoblot polyvinylidene fluoride (PVDF) membranes are a type of lab equipment used in western blotting techniques. They serve as a support medium for the transfer and immobilization of proteins separated by gel electrophoresis. PVDF membranes provide a stable and durable surface for the subsequent detection and analysis of specific proteins.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence substrate kit, by Sartorius (2 mentions)
Protein g sepharose bead, by GE Healthcare (1 mentions)
Complete edta free, by Roche (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Mab3408, by Merck Group (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Gapdh, by Merck Group (1 mentions)
β tubulin, by Merck Group (1 mentions)
Acrylamide, by Bio-Rad (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Pvdf blocking reagent for can get signal, by Toyobo (1 mentions)
Can get signal solution 1, by Toyobo (1 mentions)
Ripa buffer, by Nacalai Tesque (1 mentions)
Chemi lumi one, by Nacalai Tesque (1 mentions)
E pagel, by ATTO Corporation (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
7 protocols using immunoblot polyvinylidene fluoride pvdf membranes
1
Western Blot Analysis of Protein Interactions
2
Protein Expression Analysis by Western Blot
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Whole cell lysates were prepared from cells grown to 80-90% confluency by adding Laemmli Sample Buffer (BioRad) and collecting the lysates. The proteins in the lysates were then denatured at ∼100°C for 10 minutes. Equal amounts of protein extracts were loaded in SDS-polyacrylamide gels and subjected to electrophoresis, followed by transfer to Immunoblot polyvinylidene fluoride (PVDF) membranes (BioRad). Immunoblotting was performed using the following antibodies: EHF (5A5.5); SOX2 (D6D9, Cell signaling technologies); GAPDH (MAB374, EMD Millipore); β-Tubulin (MAB3408, EMD Millipore); AKR1C1 (CPTC-AKR1C1-1, DHSB); AKR1C2 (CPTC-AKR1C2-1, DHSB); NRF2 (D1Z9C, cell signaling technologies); ELF3 (MAB5787, R&D), SLC3A2 (15193-1-AP, Proteintech) and GPX2 (MAB5470-SP, R&D). Membranes were washed off in 0.05% Tween-20 in Tris Buffered Saline (TBS-T). The membranes were next incubated with species-specific secondary antibodies, and washed in TBS-T. The Enhanced Chemi-Luminescence (ECL) method was used to detect fractionated proteins, and the membranes were either electronically imaged using BioRad ChemiDoc imaging system (BioRad), or bioblot radiograph films (LPS).
3
Somatostatin Quantification in Cell Culture
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Fetal bovine serum (FBS) and culture media were obtained from Invitrogen (Grand Island, NY). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO). Bradford reagent, acrylamide, immunoblot polyvinylidene fluoride (PVDF) membranes, and chemoluminescent horseradish peroxidase (HRP) substrate were purchased from Bio-Rad (Madrid, Spain). Somatostatin was obtained from BCN peptides S.A. (Barcelona, Spain).
4
Western Blotting Protocol for Protein Analysis
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Western blot analysis was performed as previously described52 (link). Cells were washed three times with phosphate-buffered saline and lysed in radioimmunoprecipitation assay (RIPA) buffer (Nacalai Tesque). Cell lysates (10–20 μg) were heated in sodium dodecyl sulfate (SDS) sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 25% glycerol, 10% 2-mercaptoethanol, 0.05 mM phenylmethanesulfonyl fluoride and 0.004% bromophenol blue), separated by 10% e-PAGEL (Atto Corp, Tokyo, Japan) according to the manufacturer’s recommendations, and transferred onto immuno-blot polyvinylidene fluoride (PVDF) membranes (Bio-Rad). The membranes were blocked in PVDF Blocking Reagent for Can Get Signal (Toyobo) for 1 h at 20–25 °C and incubated with the appropriate primary antibody in Can Get Signal Solution 1 (Toyobo) overnight at 4 °C. After washing, the membranes were incubated with the secondary antibody for 1 h at 20–25 °C. The signal was visualized by Chemi-Lumi One (Nacalai Tesque) and analyzed by ChemiDoc XRS + system with Image Lab software (Bio-Rad).
5
Western Blot Analysis of Cardiac Proteins
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The cultured cardiomyocytes and heart tissues were washed twice with cold PBS, harvested, lysed in ice‐cold lysis buffer (pH = 7.4, 100 mM KCl, 10 mM HEPES, 1.5 mM MgCl), sonicated, homogenized and then subjected to protein extraction with RIRP buffer with a phosphatase inhibitor cocktail and a protease inhibitor cocktail. After centrifugation, a Pierce BCA protein assay kit was used to determine the protein concentration. Equal concentrations of soluble lysate protein (50 μg) were added to a 12% SDS‐PAGE Bis‐Tris gel, and the protein was transferred to immunoblot polyvinylidene fluoride (PVDF) membranes (Bio‐Rad). Membranes were incubated and blocked overnight at 4℃ with antibodies against p‐AMPK/AMPK, p‐Akt/Akt and GAPDH. Then, the membranes were incubated with anti‐rabbit horseradish peroxidase–conjugated secondary antibodies (1:10 000 dilution). The immunoreactive bands were analysed with an enhanced chemiluminescence substrate kit (Biological Industries, BI).
6
Western Blot Analysis of Cardiomyocytes
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Cardiomyocytes were washed twice with cold PBS, harvested, lysed in ice-cold lysis buffer (pH=7.4, 100 mM KCl, 10 mM HEPES, 1.5 mM MgCl), sonicated and homogenized in RIRP buffer. After centrifugation, a Pierce BCA protein assay kit was used to determine the protein concentration. Equal concentrations of soluble lysate protein (50 mg) were added to a 12% SDS-PAGE Bis-Tris gel, and the protein was transferred to immunoblot polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA). Membranes were incubated and blocked overnight at 4°C with antibodies against p-AMPK/AMPK, p-Akt/Akt and GAPDH. Then, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution). The immunoreactive bands were analyzed with an enhanced chemiluminescence substrate kit (Biological Industries, BI, Israel).
7
Western Blot Analysis of Protein Signaling
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Cell lysates were prepared using Mammalian Protein Extraction Reagent (Thermo Scientific) and protease and phosphatase inhibitor (Thermo Scientific). Approximately, 10 mg of protein extracted from cell lysates was separated by sodium dodecyl sulfateepolyacrylamide gel electrophoresis in a 10% polyacrylamide gel and transferred to immunoblot polyvinylidene fluoride (PVDF) membranes (Bio-Rad). Membranes were incubated with primary antibody against ERK1/2 or phosphorylated ERK1/2 (both from Cell Signaling) or CITED2, MMP-1, or MMP-13 (all from Santa Cruz), followed by incubation with secondary antibodies conjugated to horseradish peroxidase (ECL western blotting analysis system). A mouse antibody against b-actin (Sigma Aldrich) was used as a loading control.
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