Proteins from the cultured cells were extracted using radioimmunoprecipitation assay (RIPA) buffer added with a protease inhibitor PMSF (phenylmethylsulfonyl fluoride) and phosphatase inhibitor cocktail. Lysates were centrifuged at 13 000×g at 4°C for 15 minutes, and the supernatants were collected. The concentration of proteins was detected by the BCA Protein Array kit. An equal amount of protein samples were treated with 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred on to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, ISEQ00010). After blocking, the membranes were incubated with diluted primary antibody overnight at 4°C and incubated with HRP-conjugated secondary antibody for another 2 hours. After washing, the membranes were incubated with an enhanced chemiluminescence (ECL) substrate. The signals were detected by the Azure C500 Western Blot Imaging System (Azure Biosystems, Dublin, CA, USA) and then were quantified by using the ImageJ software (NIH).
Azure c500 western blot imaging system
Manufactured by Azure Biosystems
Sourced in United States
The Azure C500 Western Blot Imaging System is a compact and versatile imaging system designed for the detection and analysis of Western blot signals. It utilizes high-resolution CCD camera technology to capture images of chemiluminescent or fluorescent samples.
Automatically generated - may contain errors
Lab products found in correlation
Clarity max western ecl blotting substrate, by Bio-Rad (1 mentions)
Anti phospho mtor ser2448, by Cell Signaling Technology (1 mentions)
Anti phospho 4e bp1 thr37 46, by Cell Signaling Technology (1 mentions)
Anti 4e bp1, by Cell Signaling Technology (1 mentions)
Anti tfeb, by Fortis Life Sciences (1 mentions)
Anti phospho tfeb ser142, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase labelled secondary antibody, by Beyotime (1 mentions)
Anti mtor, by Cell Signaling Technology (1 mentions)
Gapdh, by Absin (1 mentions)
3 protocols using azure c500 western blot imaging system
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Cellular Proteins
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Western blot analysis was performed as previously described.23 The membranes were incubated at 4°C overnight with the following primary antibodies: anti‐collagen I (Abcam, ab34710), anti‐TGF‐β1 (Abcam, ab92486), anti‐fibronectin (Abcam, ab23750), anti‐LC3B (Sigma, L7543), anti‐p62/SQSTM1 (Abcam, ab91526), anti‐Cathepsin B (Abcam, ab58802), anti‐phospho‐mTOR (Ser2448) (Cell Signaling, 5536S), anti‐mTOR (Cell Signaling, 2983S), anti‐phospho‐4E‐BP1(Thr37/46) (Cell Signaling, 2855S), anti‐4E‐BP1 (Cell Signaling, 9644S), anti‐phospho‐TFEB (Ser142) (Millipore, ABE1971‐I), anti‐TFEB (BETHYL, A303‐672). β‐actin (Santa Cruz, sc‐47778) and GAPDH (Absin Bioscience, abs132004) were used as the loading control in vivo and in vitro experiments, respectively. The membranes were washed with PBS‐T five times and then incubated with horseradish peroxidase‐labelled secondary antibody (Beyotime Biotechnology, China) for 1 h at room temperature. After visualized with Clarity Max™ Western ECL Blotting Substrates (Bio‐Rad, Hercules, CA, USA), the Azure C500 Western Blot Imaging System (Azure Biosystems, Dublin, CA, USA) was used to detect the immunoreactive signals. The optical density of each band was then quantified using ImageJ software (NIH).
3
Renal Protein Expression Analysis
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Western blots were used to detect renal expression of target proteins. Protein samples were subjected to 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), then transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% BSA in TBST solution, incubated with the primary antibody, washed and then incubated with HRP-conjugated secondary antibody. Chemiluminescent reactions with luminol were visualised in the Azure C500 Western Blot Imaging System and then analysed in Image J.
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