Bca protein kit

The clotted plasma sample was centrifuged at 2000 × g at 4 °C for 20 min before assays. Liver sample was defrosted and homogenized on ice with 10 volumes of cold buffer consisting of 250 mmol L⁻¹ sucrose, 5 mmol L⁻¹ Tris–HCl, and 0.1 mmol L⁻¹ edetic acid–2Na (pH 7.5). The homogenate was centrifuged at 4000 × g at 4 °C for 15 min to obtain the supernatant for biochemical assays. The plasma and homogenate were used to determine the content of malondialdehyde (MDA), oxidized glutathione (GSSG), reduced glutathione (GSH), enzymatic activity of catalase (CAT), total superoxide dismutase (T-SOD), CuZn superoxide dismutase (CuZn-SOD), glutathione peroxidase (GP_X) and total antioxidative capability (T-AOC) using commercial assay kits (Nanjing Jiancheng Institute, Jiangsu, China). Protein concentration of liver tissue was determined using bicinchoninic acid (BCA) as a detection reagent for Cu⁺ following the reduction of Cu²⁺ by protein in an alkaline environment (BCA protein kit, Sangon company, China). All samples were measured in duplicate.

Superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) belong to the antioxidant enzyme system. Malondialdehyde (MDA) is considered the most common lipid peroxidation marker. The four enzyme markers indicate the extent of algal cell oxidative stress and damage (Sun et al., 2019 (link); Zheng et al., 2021 (link)). Following the manufacturer’s instructions, these indexes were determined using several kits (Shanghai Enzyme-linked Biotechnology Co., Shanghai, China). The enzyme activities were analysed and expressed as protein levels. Protein concentrations were determined using bicinchoninic acid (BCA) as the detection reagent for Cu⁺ following the reduction of Cu²⁺ by protein in an alkaline environment (BCA protein kit, Sangon company, China). Measurements were made using a microplate reader using a microwell plate protocol A590 in the manufacturer’s instructions.

β-tubulin III and GFAP protein expressions of differentiated NSCs on hydrogels with and without ES were also quantified using the Western blot analysis. The total protein concentration of cell lysates was measured by the BCA method using the BCA protein kit (Sangon Biotech, Shanghai, China). Forty micrograms of proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Sango Biotech, Shanghai, China). The membranes were blocked with 5% non-fat milk for 2 h and incubated with primary antibodies against β-tubulin III (1:100; Abcam, Shanghai, China), GFAP (1:200; Abcam, Shanghai, China), and GAPDH (1:1000; Abcam, Shanghai, China). After incubation for 3 h at room temperature, followed by 4°C overnight, the membranes were incubated with secondary antibody (Beyotime Institute of Biotechnology, Shanghai, China) at room temperature for 1 h and visualized using the ECL system (GE Healthcare Bio-Sciences AB, Image Quant LAS 500, Sweden).