Chondrocytes were washed with PBS three times and lysed with RIPA supplemented with 1 mM protease inhibitor cocktail and 1 mM phosphatase inhibitor cocktail (Boster, Wuhan, China). Twenty five micrograms protein samples were separated on SDS-polyacrylamide gels and transfered onto a PVDF membrane. The membrane was firstly blocked with 5% bovine serum albumin (BSA) for 1 h and then incubated overnight with primary antibodies at 4°C. Subsequently, blots were washed with Tris-buffered saline with 0.1% Tween-20 (TBST) three times and incubated with horse-radish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Finally, the immunoreactive bands were detected with the Western ECL System (Boster, Wuhan, China). GAPDH was used as the loading control and representative bands were shown. Western blots were repeated at least three times.
Western ecl system
Manufactured by Boster Bio
Sourced in United States
The Western ECL System is a laboratory equipment used for the detection and visualization of proteins in Western blot analysis. It provides a chemiluminescent detection method that enables the quantification of target proteins.
Automatically generated - may contain errors
2 protocols using western ecl system
1
Chondrocyte Protein Extraction and Western Blot
2
Protein Extraction and Western Blot Analysis
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Chondrocytes from each treatment group were lysed with radioimmunoprecipitation assay (RIPA) (Boster) buffer, which contains 1% protease inhibitor and 1% phosphatase inhibitor for 15 minutes on ice. Then, lysed protein samples were gathered and centrifuged (12,000 g, 30 minutes, 4°C). After that, a bicinchoninic acid (BCA) assay kit (Boster) was employed to quantify the protein content of each sample after collecting the supernatant after centrifugation. In electrophoresis on a polyacrylamide-sodium dodecyl sulfate gel (SDS-PAGE), multiple proteins were isolated and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Next, the membranes were incubated with 5% skim milk for one hour at room temperature to inhibit any potential permeability, and treated with primary antibodies for more than 14 hours at 4°C, followed by one hour of treatment with secondary antibodies at room temperature. Protein bands were visualized using a Western ECL System (Boster), and images were captured using a Bio-Rad scanner (USA).
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