The treated cells were lysed with the radio immunoprecipitation assay (RIPA; Applygen, China) reagent on ice for 30 min, and then the mixture was centrifugated at 15000 g and 4 °C for 5 min to obtain the supernatant. The protein concentration was determined and compared to the standard BSA curve. After that, 20 μg of proteins were separated via 10% SDS-PAGE and then transferred to the polyvinylidine fluoride (PVDF) membrane (Absin, China). Next, the membrane was blocked with 1% skim milk for 30 min and then reacted with the required primary antibodies purchased from CST: anti-caspase-3 (14220S, 1:2000), anti-cleaved caspase-3 (9579S, 1:3000), anti-caspase-9 (20750S, 1:5000), anti-cleaved caspase-9 (20750S, 1:1000), anti-LC3 II/I (12741S, 1:5000), anti-Beclin-1 (3495S, 1:4000), anti-ULK1 (8054S, 1:3000) and anti-β-actin (4970S, 1:10000) at 4 °C overnight. On the second day, the PVDFs were rinsed with Tris-buffered saline with Tween-20 and further incubated with the secondary antibody (14708S, 1:15000) for 1 h. At last, the enhanced chemiluminescence reagent (Abbkine, USA) was used for developing, and ImageJ was used for gray analysis.
Pvdf membrane
Manufactured by Absin
Sourced in China
PVDF membrane is a type of laboratory filtration membrane made from polyvinylidene fluoride (PVDF) material. It is known for its chemical and temperature resistance properties. The core function of the PVDF membrane is to act as a filtration medium for various applications in research and industrial laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence reagent, by Abbkine (1 mentions)
Radioimmunoprecipitation assay (ripa), by Applygen (1 mentions)
Ab39012, by Abcam (1 mentions)
Aggrecan, by Abcam (1 mentions)
Ab36861, by Abcam (1 mentions)
Ab53015, by Abcam (1 mentions)
Ab84792, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab111523, by Abcam (1 mentions)
Ab34712, by Abcam (1 mentions)
Ecl detection kit, by Absin (1 mentions)
Bca protein assay kit, by Takara Bio (1 mentions)
Ab8227, by Abcam (1 mentions)
Protease inhibitor mixture, by Thermo Fisher Scientific (1 mentions)
Ab8226, by Abcam (1 mentions)
Ripa buffer, by Abcam (1 mentions)
Bca protein assay kit, by Sangon (1 mentions)
Bovine serum albumin (bsa), by Yeasen (1 mentions)
Ecl luminescence reagent, by Absin (1 mentions)
Bca protein concentration assay kit, by Biosharp (1 mentions)
4 protocols using pvdf membrane
1
Protein Extraction and Western Blot Analysis
2
Protein Quantification and Immunoblotting Analysis
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Total protein was extracted using the Tractor Buffer Kit (Takara), and the concentration of protein was examined using the BCA protein assay Kit (Takara). Then, equal amount of protein was isolated by 12% SDS-PAGE and transferred into PVDF membrane (Absin). The membranes containing proteins were blocked with 5% non-fat milk for 1 h and then probed with the primary antibodies against Aggrecan (ab36861; Abcam, Cambridge, MA, USA), COL2 (ab34712; Abcam), MMP13 (ab39012; Abcam), MMP3 (ab53015; Abcam), ADAMTS4 (ab84792; Abcam), SPRY1 (ab111523; Abcam) and β-actin (ab8227; Abcam) for 16 h at 4 °C. Next, the membranes containing proteins were probed with the goat anti-rabbit secondary antibody (ab205718; Abcam) for 1.5 h at room temperature. Finally, protein blot signals were visualized using the enhanced chemiluminescence (ECL) detection kit (Absin).
3
Western Blot Analysis of uMtCK Protein
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Cells were homogenized and total protein was obtained using RIPA buffer (Abcam, Cambridge, UK) containing protease inhibitor mixture (ThermoFisher Scientific, Waltham, MA, USA). Protein concentration was assessed with BCA Protein Assay Kit (Sangon, Shangai, China). Fifty µg of protein of each lysate was electrophoresed through 10% SDS-PAGE and then transferred to PVDF membrane (Absin, Shanghai, China). Membranes were blocked with 2% BSA (Yeasen, Shanghai, China) and then labeled with primary antibodies against uMtCK (1:1000, PA5-96224; ThermoFisher Scientific, Waltham, MA, USA) and β-actin (1:2000, ab8226; Abcam, Cambridge, UK) at 37 °C for 90 min. Membranes were subsequently rinsed with PBST, and then incubated with HRP-conjured secondary antibody (1:1000; Beyotime, Jiangsu, China). Immunoblots were visualized using ECL luminescence reagent (Absin, Shanghai, China).
4
Western Blot Analysis of BPTC Proteins
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The BPTCs treated with glucose with or without the addition of 100 nmol/L rapamycin for 24 h were collected for western blotting analysis. The BPTCs were washed three times with PBS, and 1 mL of cell RIPA solution was added to obtain total protein. The protein concentration was detected by the BCA protein concentration assay kit (Biosharp, Guangzhou, China). Western blot analysis was performed in brief as follows: 20 µg of protein/well was separated on 6–15% separating gel, 2–10% concentrating gel (Servicebio, Wuhan, China) and transferred to PVDF membrane (Absin, Shanghai, China). The membranes were sequentially closed with 5% skimmed milk generated in Tris-buffer, followed by incubation using primary antibodies, overnight at 4 °C. The membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody for 1 h at room temperature. Subsequently, the membranes were incubated with the specific ultrasensitive ECL chemiluminescent substrate (Biosharp, Guangzhou, China) and visualization of the proteins was achieved with the ChemiDOC MP (Bio-Rad). The mean values of protein in 1 mg/mL treatment were set to 1.00. Grayscale analysis was performed using ImageJ Software 1.8.0. The complete details of primary antibodies are listed in Table 2 .
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