Cells and clinical tissues were lysed for 30–45 mins on ice in lysis buffer containing freshly added protease inhibitor cocktail (Roche Diagnostics, USA). After BCA quantification (Pierce, USA), the protein was added to 5× loading buffer and boiled at 95°C for 10 min. Equal amounts of protein were separated by SDS-PAGE and blotted onto activated polyvinylidene difluoride membranes (Millipore, USA). After blocking, the membranes were incubated with primary antibodies overnight at 4°C. The antibodies used were as follows: BMX (1:2000, 610793, BD), phospho-Etk (Tyr40) (1:500, #3211, Cell Signaling Technology), AKT1 (1:500, sc-5298, Santa Cruz Biotechnology), p-AKT (1:1000, #4060, Cell Signaling Technology), mTOR (1:1000, A2445, ABclonal), p-mTOR (1:1000, AP0094, ABclonal), STAT3 (1:1000, #9132, Cell Signaling Technology), phospho-STAT3 (Tyr705) (1:1000, #4113, Cell Signaling Technology), β-actin and GAPDH (1:1000, Santa Cruz Biotechnology). Blots were incubated with secondary antibodies coupled to horseradish peroxidase (Thermo Fisher Scientific, USA) for 1 h and visualized using ECL detection (Millipore) on X-ray film. Relative quantitation was analyzed with Quantity One software.
Activated polyvinylidene difluoride membranes
Manufactured by Merck Group
Sourced in United States
Activated polyvinylidene difluoride membranes are a type of laboratory equipment used for various scientific and analytical applications. These membranes are made of polyvinylidene difluoride (PVDF), a polymer material with chemical and physical properties that make it suitable for numerous laboratory tasks. The activation process enhances the membrane's ability to interact with and immobilize specific biomolecules, such as proteins, peptides, or nucleic acids. These membranes provide a versatile platform for techniques like western blotting, protein transfer, and affinity-based separations.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Cell Signaling Technology (2 mentions)
Secondary antibodies coupled to horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
P mtor, by ABclonal (1 mentions)
Enhanced chemiluminescence detection, by Merck Group (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence detection system, by Thermo Fisher Scientific (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
Trans blot, by Bio-Rad (1 mentions)
3 protocols using activated polyvinylidene difluoride membranes
1
Western Blot Analysis of Cell Signaling
2
Western Blot Protein Analysis Protocol
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Cell lysates were extracted in RIPA buffer (Pierce) and quantified by a BCA protein assay kit (Pierce) according to the manufacturer's protocol. Equal amounts (30 μg) of total protein were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto activated polyvinylidene difluoride membranes (Millipore). After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies, as listed in Supplementary Table S2 . The blots were then incubated with secondary antibody conjugated with horseradish peroxidase and immunoreacted bands were detected by enhanced chemiluminescence detection (Millipore).
3
Quantitative Analysis of Atrial Proteins
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Whole-cell lysates from frozen atrial tissue were run simultaneously to ensure identical conditions. From each sample, 60 mg total protein was loaded on a 4% to 12% gradient sodium dodecyl sulfate polyacrylamide electrophoresis gel (Life Technologies, Grand Island, NY). The proteins were transferred to activated polyvinylidene difluoride membranes (Millipore, Billerica, MA) with a semidry transfer cell apparatus (Trans-Blot; Bio-Rad, Hercules, CA). Each membrane was incubated with specific primary antibodies overnight at 4 C and secondary antibodies for 1 hour at room temperature. Glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology, Danvers, MA) was used to normalize the protein loading.
JAK2, suppressor of cytokine signaling 3 (SOCS3), p-STAT1/3/5, STAT1/3/5, apoptotic markers p-Bc12, Bc12, caspase 3, Mcl-1, Bcl-xL, and p-Akt (Cell Signaling Technology), and oxidative stress markers nuclear factor (NF)-kB (Abcam, Cambridge, United Kingdom) and manganese-containing superoxide dismutase (R&D Systems, Minneapolis, MN) were probed. Neurotrophin-3 (Abbiotec, San Diego, CA) and nerve growth factor (Santa Cruz Biotechnology) were examined as markers for neurogenesis. Immune complexes were visualized with an enhanced chemiluminescence detection system (Thermo Scientific, Amersham, Piscataway, NJ).
JAK2, suppressor of cytokine signaling 3 (SOCS3), p-STAT1/3/5, STAT1/3/5, apoptotic markers p-Bc12, Bc12, caspase 3, Mcl-1, Bcl-xL, and p-Akt (Cell Signaling Technology), and oxidative stress markers nuclear factor (NF)-kB (Abcam, Cambridge, United Kingdom) and manganese-containing superoxide dismutase (R&D Systems, Minneapolis, MN) were probed. Neurotrophin-3 (Abbiotec, San Diego, CA) and nerve growth factor (Santa Cruz Biotechnology) were examined as markers for neurogenesis. Immune complexes were visualized with an enhanced chemiluminescence detection system (Thermo Scientific, Amersham, Piscataway, NJ).
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