Frozen tissue samples were homogenised in RIPA buffer consisting of 1% protease inhibitor mixture. The mixture was centrifuged at 12 000 g for 15 min at 4 °C and the supernatant was obtained. Total proteins were quantified. Per sample, 30 μg of protein was separated onto a denaturing polyacrylamide gel containing SDS and transferred to a methanol-activated PVDF filter membrane (Bio-Rad, Hercules, CA, USA). Before immunodetection, membranes were blocked with 5% non-fat dry milk. Primary antibodies, anti-PGK1 (1:500; rabbit polyclonal; Abcam, Cambridge, MA, USA) were diluted in the buffer and incubated at 4 °C overnight. After subsequently washing with TBST, the membranes were incubated with secondary antibody (horseradish peroxidase-conjugated anti-rabbit) for 1 h at room temperature. The experiment was repeated in triplicate. The bands were detected by enhanced chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China).
Pvdf filter membrane
Manufactured by Bio-Rad
Sourced in United States
The PVDF filter membrane is a highly durable and chemically resistant material commonly used in various laboratory applications. It acts as a physical barrier, allowing the passage of liquids and gases while retaining specific molecules or particles based on their size. The PVDF membrane is inert, thermostable, and offers excellent mechanical strength, making it a versatile choice for filtration, separation, and purification processes in research and industrial settings.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti pgk1, by Abcam (1 mentions)
Enhanced chemiluminescence detection reagent, by Applygen (1 mentions)
Enhanced chemiluminescence reaction kit, by Applygen (1 mentions)
Ripa lysis buffer, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti smad1, by Abcam (1 mentions)
Anti p smad1, by Abcam (1 mentions)
Anti smad3, by Abcam (1 mentions)
Anti p smad3, by Abcam (1 mentions)
Anti tgf β1, by Abcam (1 mentions)
2 protocols using pvdf filter membrane
1
Western Blot Analysis of PGK1 Protein
2
Western Blot Analysis of BZW1, Smad, and TGF-β1
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Proteins were extracted by radioimmunoprecipitation assay (RIPA) Lysis buffer (Abcam, Cambridge, MA, USA) and quantified by bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). 40 µg of protein per sample was separated onto 10% SDS-PAGE gels and transferred onto polyvinyl difluoride (PVDF) filter membrane (Bio-Rad, Hercules, CA, USA). Then, PVDF membranes were blocked with 5% non-fat dried milk for 2 h, and incubated with anti-BZW1 (1: 1000, ABcam Corp, USA), anti-Smad1 (1: 1000, ABcam Corp, USA), anti-p-Smad1 (1: 1000, ABcam Corp, USA), anti-Smad3 (1: 1000, ABcam Corp, USA), anti-p-Smad3 (1: 1000, ABcam Corp, USA), anti-TGF-β1 (1: 2000, ABcam Corp, USA) and anti-GAPDH (1: 2000, ABcam Corp, USA), respectively at 4 overnight. Finally, membranes were incubated with secondary antibodies (horseradish peroxidase-conjugated anti-rabbit). Signals were developed using an enhanced chemiluminescence reaction kit (Applygen Technologies, Beijing, China).
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