Horseradish peroxidase hrp labeled secondary antibody

H₂O₂-treated BmN-SWU1 cells from each treatment were collected by centrifugation and added to radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime) on ice. After centrifugation at 13,000 g for 5 min at 4°C, the supernatants were extracted and mixed with sodium dodecyl sulfate (SDS) loading buffer. Total protein concentration was estimated using a bicinchoninic acid (BCA) Protein Assay Kit (Beyotime) as described by the manufacturer. Equal amounts of proteins were separated on a 10% SDS polyacrylamide gel electrophoresis (PAGE) and finally transferred onto polyvinylidene difluoride (PVDF) membranes using standard protocols. The membranes were then incubated with the following primary antibodies: anti-cytochrome c (1:500; Beyotime), anti-Bcl-2 (1:1000; Cell Signaling Technology, Danvers, MA, USA) and anti-P53 (1:1000; Cell Signaling Technology), followed by incubation with the corresponding horseradish peroxidase (HRP)-labeled secondary antibody (1:5000; Beyotime). Protein bands were visualized using the Lumi-Light PLUS Western blotting substrate (Roche, Mannheim, Germany). Tubulin (Beyotime) was used as the loading control.

Equivalent proteins were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto polyvinylidene fluoride (PVDF) membranes, followed by the incubation with blocking solution containing phosphate-buffered solution (PBS) (pH 7.4) with 5% skimmed milk and 0.1% Tween-20 for 1 h at 37 °C, and then with an anti-MMAE primary antibody (1:1000 dilution) for 2 h at 37 °C. Blots were then washed three times with 0.1% Tween-20/PBS (TPBS), incubated with a 1:1000 dilution of horse radish peroxidase (HRP)-labeled secondary antibody (Beyotime, Shanghai, China) for 2 h at 37 °C and washed three times with TPBS again. Protein bands were visualized with DAB Horseradish Peroxidase Color Development Kit (Beyotime, Shanghai, China).