Detection of the COL2A1 protein by western blotting was carried out according to [46 (link)]. Briefly, equal amounts of total protein (30 µg) from different synovial fluid samples were loaded for each lane and separated by 10% SDS-PAGE, followed by electroblotting by tank transfer technique to the PVDF membrane. The primary antibody used was polyclonal anti-type II collagen, COL2A1 antibody (Chongqing Biospes, cat# YPA1669), and HRP-conjugate goat anti-rabbit IgG as a secondary antibody (Chongqing Biospes, cat#BSA1013). The desired specific bands were developed by using DAB horseradish peroxidase chromogenic kit (Chongqing Biospes, cat#BWR1069).
Both zymogram gels and western blot membranes were digitally scanned in the true color. The bands’ intensities were quantified using my Image analysis software v2.0 (Thermo scientific) after being conversed with the grayscale. For western blot analysis, COL2A1 blots were normalized to the total protein according to Fosang et al. [47 (link)].
Both zymogram gels and western blot membranes were digitally scanned in the true color. The bands’ intensities were quantified using my Image analysis software v2.0 (Thermo scientific) after being conversed with the grayscale. For western blot analysis, COL2A1 blots were normalized to the total protein according to Fosang et al. [47 (link)].