Pierce enhanced chemiluminescence ecl reagents

Protein was extracted from cells 3 d after transfection, as follows. The cells were washed twice with phosphate-buffered saline pre-chilled to 4 °C. Then, 100 μL NP-40 lysis buffer (Beyotime, Shanghai, China, P0013F) and 1 mM phenylmethylsulfonyl fluoride (PMSF, YEASEN, Shanghai, China, 20104ES03) were added to wash off the adherent cells. The samples were centrifuged at 13,000× g for 5 min at 4 °C, and the supernatant was transferred to a new 1.5-mL centrifuge tube pre-chilled at 4 °C. The collected cell lysate was immediately assayed by Western blotting or stored at −20 °C.
After determining cell lysate concentration using a BCA Protein Assay Kit (Beyotime, Shanghai, China, P0012), 50 μg protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membrane (PVDF, GE Healthcare, Freiburg, Germany). The following primary antibodies were used: anti-DsRed antibody (Biovision, Tucson, AZ, USA, 3993-100) and anti-GFP (Biovision, 3999-100). The following secondary antibodies were used: Goat anti-Rabbit IgG (H + L) Secondary Antibody (Invitrogen, Carlsbad, CA, USA, 31460), and Goat anti-Mouse IgG (H + L) Secondary Antibody (Invitrogen, 31430). The protein signal was detected by Pierce enhanced chemiluminescence (ECL) reagents (Thermo Fisher Scientific, Waltham, MA, USA, 32106).

Forty-eight hours after transfection, proteins were extracted from BmE cells with NP-40 lysis buffer (Beyotime, Shanghai, China) containing the protease inhibitor phenylmethanesulfonyl fluoride (PMSF). The protein concentration was measured using the BCA assay (Beyotime, Shanghai, China). Protein samples (20 μg) were separated using 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein gel, and then transferred to a polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Pittsburgh, USA). Subsequently, samples were treated with primary antibodies (Cas9/tubulin-specific antibody, 1:10,000) for 120 min at 37 °C before incubation for 60 min at 37 °C with a horseradish peroxidase (HRP)-coupled secondary sheep anti-mouse IgG antibody (1:20,000). Finally, the signal was detected with Pierce enhanced chemiluminescence (ECL) reagents (Thermo Fisher Scientific, New York, NY, USA).