Bca protein quantitative kit

The extraction of total proteins was conducted from neurons and tissues around the diseased cortex in rats, and the protein concentration was measured using a Bicinchoninic acid (BCA) protein quantitative kit (Epizyme, Shanghai). The protein samples were evenly divided and subjected to polyacrylamide gel electrophoresis. Subsequently, they were placed onto polyvinylidene fluoride (PVDF) membranes and subsequently treated with 5% skim milk to prevent non-specific binding for 2 h at ambient temperature. The PVDF membranes were subjected to incubation overnight at 4°C with primary antibodies (Abs) targeting HDAC3, H3, H4, acetyl-H3, acetyl-H4 (Abcam, UK), Nrf2, HO-1, Neuron Specific Enolase (NSE), Caspase1, IL-1β, HMGB1, TLR4, BCL2-Associated X (BAX), B-cell lymphoma-2 (BCL-2), β-actin (Proteintech, Wuhan), NQO1 (Bioworld, UK) and Ionized calcium bindingadaptor molecule-1 (Iba-1)(Cell Signaling Technology, USA). The PVDF membranes were then treated with the matching secondary Abs linked to Horseradish Peroxidase (HRP) (Epizyme, Shanghai) for 1 h at ambient temperature on the next day. Protein bands were visualized by employing enhanced chemiluminescence (Epizyme, Shanghai), and the optical density of protein strips was measured using Image J software.

First, the cell lysate was prepared (1 ml working solution = 1 ml RIPA (EpiZyme, cat. no. PC101) + 10ul Protease/Phosphatase Inhibitor Cocktail (100 × ; CST, cat. no. 5872S)) and placed on ice for later use. Chop the fresh tissues into fragments with a diameter of about 1–2 mm, grind them into powder, add 500ul cell lysis solution, and thoroughly mix. Leave it on ice for 30 min to allow the cells to break down sufficiently. Centrifuge in high speed centrifuge, centrifuge conditions: 4 °C, 14000 rpm, 15 min, supernatant was collected, the total concentration of extracted protein was detected by BCA protein quantitative kit (EpiZyme, cat. no. ZJ101), and stored at -80℃ for later use.