Horseradish peroxidase hrp conjugated igg

The proteins, extracted from human or mice skin samples as well as mice skin fibroblasts, were separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and transferred to a polyvinylidene fluoride (PVDF) membrane. Then, the membranes were immerged into 5% milk without fat to block for 2 hours. Next, they were incubated with primary antibodies including anti‐SIRT3, caspase 3, MLKL, p‐MLKL, RIPK1 (1:1000, Cell Signaling Technology), anti‐RIPK3 (1:1000, Novusbio), anti‐α‐SMA (1:1000, Bosterbio), anti‐GAPDH (1:5000, Sigma‐Aldrich) and anti‐β‐tubulin (1:3000, CMCTAG) at 4°C for more than 12 hours. Horseradish peroxidase (HRP)‐conjugated IgG (Beyotime) or enhanced chemiluminescence (ECL, Thermo Fisher Scientific Inc) was applied to visualize the protein bands.

The proteins of about 50 μg were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. Next, the membrane was blocked by 5% milk without fat for 2 h followed by incubation with primary antibodies (Table 2) [45 (link)–47 (link)] at 4°C overnight. After washing, the membrane was incubated by horseradish peroxidase- (HRP-) conjugated IgG (Beyotime, Shanghai, China) for another 2 h. Enhanced chemiluminescence (ECL, Thermo Fisher Scientific Inc., Rockford, IL, USA) was dropped on the membrane to visualize the protein bands.