Western blotting was conducted following the method of Ozaki et al.[32] (link). Briefly, each sample was separated using SDS-PAGE and transferred onto Immobilon-P polyvinylidene fluoride (PVDF) transfer membranes (catalog number: IPVH00010; EMD Millipore, Billerica, MA, USA). The membranes were blocked with blocking buffer (10 mM sodium phosphate buffer, pH 7.4, 0.14 M NaCl, and 0.05% Tween 20 [TW-PBS] containing 1% skim milk or TW-PBS containing 5% skim milk) for 4 h at 25°C and then incubated overnight with the primary antibodies at 4°C. After washing with TW-PBS, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies overnight at 4°C. Each antibody had been diluted with the blocking buffer as follows: anti-mouse ES1 (1:1000), anti-porin (1:1000), anti-Ak2 (1:2000 or 1:1000), anti-COX4 (1:2000), anti-AIF (1:500), anti-ATP5A (1:200), and anti-beta-actin (1:10,000). After incubation and washing, the immunoreactive signals were detected using the ECL Prime Western Blotting Detection Kit (catalog number: RPN2236; Cytiva, Tokyo, Japan). The images were captured using a luminescent image analyzer (ChemiDoc XRS Plus; catalog number: 1708265J1PC; Bio-Rad Laboratories, Hercules, CA, USA).
Immobilon p polyvinylidene fluoride pvdf transfer membrane
Manufactured by Merck Group
Sourced in United States
Immobilon-P polyvinylidene fluoride (PVDF) transfer membranes are a type of laboratory equipment used for the transfer and immobilization of proteins, nucleic acids, and other biomolecules from electrophoretic gels onto a solid support for further analysis. These membranes provide a high-binding capacity and are compatible with a variety of detection methods.
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Lab products found in correlation
Micro bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Precision plus protein kaleidoscope standard, by Bio-Rad (2 mentions)
P5726, by Merck Group (2 mentions)
Luminata classico, by Merck Group (2 mentions)
P7626, by Merck Group (2 mentions)
Chemidoc xrs plus, by Bio-Rad (1 mentions)
Ecl prime western blotting detection kit, by Cytiva (1 mentions)
Anti β actin monoclonal antibody, by Merck Group (1 mentions)
Metformin 1 1 dimethylbiguanide hydrochloride, by Merck Group (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Pierce enhanced chemiluminescence ecl detection kit, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg fab specific peroxidase antibody, by Merck Group (1 mentions)
Anti rabbit igg whole molecule peroxidase antibody, by Merck Group (1 mentions)
P8340, by Merck Group (1 mentions)
Luminata crescendo western horseradish peroxidase hpr substrates, by Merck Group (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Luminata crescendo, by Merck Group (1 mentions)
4 protocols using immobilon p polyvinylidene fluoride pvdf transfer membrane
1
Western Blot Technique for Protein Analysis
2
Metformin-Based Protein Analysis Protocol
3
Protein Extraction and Western Blot Analysis
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Following treatment, cells were lysed on ice in a buffer consisting of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton x-100, 1% sodium deoxcycholate, 0.1% SDS, phosphatase inhibitor (P5726, Sigma Aldrich), protease inhibitor (P8340, Sigma Aldrich), and phenylmethylsulfonyl fluoride (PMSF, P7626, Sigma Aldrich) for 30 minutes. Lysates were centrifuged at 14 000 rpm for 30 minutes at 4°C. Protein concentrations were determined using a Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific). Proteins were separated on SDS-PAGE gels by electrophoresis and transferred to Immobilon®-P polyvinylidene fluoride (PVDF) transfer membrane (EMD Millipore). Precision Plus Protein Kaleidoscope Standards (161–0375, Bio-Rad, Hercules, CA) molecular weight markers were used to confirm expected size of target proteins. Antibodies were used per the manufacturers’ recommended protocol. Samples were visualized by enhanced chemiluminescence (ECL) using Luminata Classico or Luminata Crescendo Western horseradish peroxidase (HPR) substrates (EMD Millipore). Anti-β-actin or vinculin served as an internal control to ensure equal protein loading.
4
Protein Extraction and Western Blot Analysis
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Cells or homogenized tumor specimens were lysed on ice in a buffer consisting of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton x-100, 1% sodium deoxcycholate, 0.1% SDS, phosphatase inhibitor (P5726, Sigma Aldrich), protease inhibitor (P8340, Sigma Aldrich), and phenylmethylsulfonyl fluoride (PMSF, P7626, Sigma Aldrich) for 30 min. Lysates were centrifuged at 17,000× g for 30 min at 4 °C. Protein concentrations were determined using a Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific). Proteins were separated on SDS-PAGE gels by electrophoresis and transferred to Immobilon®-P polyvinylidene fluoride (PVDF) transfer membrane (EMD Millipore, Burlington, MA, USA). In order to confirm the expected size of target proteins, Precision Plus Protein Kaleidoscope Standards (161-0375, Bio-Rad) molecular weight markers were used. Antibodies were used per the manufacturers’ recommended protocol. Luminata Classico or Luminata Crescendo (EMD Millipore) substrates were used to visualize immunoblots by enhanced chemiluminescence (ECL) of horseradish peroxidase (HPR)-conjugated secondary antibodies. β-actin or vinculin served as a control to ensure equal protein loading. We performed densitometry of Western blots using ImageJ software (Ver 1.49, http://imagej.nih.gov/ij (accessed on 7 July 2018).
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