The cultured cells and tumor tissues were collected and frozen at −80 °C, and total proteins were extracted using RIPA Lysis Buffer (Beyotime, Shanghai, China) and quantified with the Coomassie Plus (Bradford) Assay Kit (Thermo Scientific, Cat#23236). Western blot analysis was performed according to previous studies.27 (link) After separation through a precast BIS-Tris Gel (4–20%, Cat#abs9384, Absin, Shanghai, China), proteins were transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk for 45 min at room temperature and then incubated waveringly with primary antibodies, including anti-HMGB1 (1:1000), anti-GAPDH (1:20,000), anti-Bcl-2 (1:2000), anti-cleaved-Caspase-3 antibody (1:1000), and anti-AR antibody (1:100) at 4 °C overnight. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies (1:10,000) at room temperature for 1 h. Finally, the bands were visualized by the ECL method using a ChemiScope 6000 imaging system (Clinx Science Instruments, China) and quantified using ImageJ software. The assay was independently repeated three times.
Chemiscope 6000 imaging system
Manufactured by Clinx
Sourced in China
The ChemiScope 6000 is a high-resolution imaging system designed for scientific and laboratory applications. It features a 6-megapixel camera and advanced optics to capture detailed images of samples. The system is capable of acquiring images in various modes, including brightfield, fluorescence, and chemiluminescence. The ChemiScope 6000 is a versatile tool for a range of applications, including protein analysis, gel documentation, and molecular biology research.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Promega (1 mentions)
Westernbright ecl reagent, by Advansta (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
2 protocols using chemiscope 6000 imaging system
1
Quantitative Western Blot Analysis of Proteins
2
Analyzing cGAS and TBK1 in BV-2 Cells
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Cell lysates were extracted from BV-2 cells using RIPA lysis buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors (Roche, Germany). The lysates were then run on 10% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (Millipore, USA). Primary antibodies used for Western blotting included monoclonal rabbit anti-cGAS (#31659S, Cell Signaling Technology, USA), monoclonal rabbit anti-phospho-tank binding kinase 1 (TBK1)/NAK (Ser172) (#5483S, Cell Signaling Technology, USA) and monoclonal rabbit anti-TBK1/NAK (#ab40676, Abcam, USA). Following incubation with the primary antibodies, horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Promega, USA) and the WesternBright ECL reagent (Advansta, USA) were sequentially applied. The blots were scanned using a ChemiScope 6000 imaging system (Clinx Science Instruments, China). Densitometry analysis was conducted using ImageJ.
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