Clarity western ecl substrate for hrp

Overexpression efficiency was measured by western blot analysis under standard conditions, using a nitrocellulose membrane and horseradish peroxidase (HRP)-conjugated secondary antibodies. Semi-quantitative measurement of protein levels was performed using ImageLab 5.1 software (Bio-Rad, Hercules, California, USA). The chemiluminescent signal was detected using the Clarity™ Western ECL Substrate for HRP (Bio-Rad, 1705060, Hercules, California, USA).

10⁷ cells were lysed in 100 µl of RIPA buffer with protease inhibitor cocktail and EDTA (Thermo Fisher Scientific). Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins were separated on 4–15% Mini-PROTEAN TGX Stain-free Gel (Bio Rad). After electrophoresis, proteins were transferred onto 0.45 µm PVDF Low Fluorescence membrane (Bio Rad, Hercules, CA, USA). Membranes were blocked using 5% non-fat milk and incubated with 1:2000 mouse anti-Cas9 antibody (#14697, Cell Signaling Technology, Danvers, MA, USA) or 1:2000 rabbit anti-BIM antibody (#2933, Cell Signaling Technology). After washing, membranes were incubated with horseradish peroxidase (HRP) conjugated with 1:10,000 anti-mouse (A9917, Sigma Aldrich) or 1:40,000 anti-rabbit (ab97051, Abcam, Cambridge, U) secondary antibody. Immunoreactive protein bands were detected with Clarity Western ECL Substrate for HRP (Bio-Rad) on Chemidoc Imaging System (Bio Rad). The abundance of target protein was assessed in reference to the total protein on a blot in Stain-Free technology using Image Lab 6.0.1 software (Bio Rad). Each experiment was conducted in three biological replicates.