Quick block western reagent

The E. coli cells were collected and washed twice with phosphate buffered saline (PBS, 0.01 M, pH 7.2–7.4) and disrupted using a high-pressure cell disruptor (Constant Systems LTD, UK). The his₆-tagged recombinant protein was purified using Ni-NTA His·Bind Column (Novagen). The protein was quantified using a Bradford protein assay kit (Beyotime, China), fractionated on a 12% SDS-PAGE gel and visualized by Coomassie blue staining. For western blot, proteins were transferred to PVDF membrane (Millipore) and blocked using quick block western reagent (Beyotime, China). HRP Anti-6X His tag antibody [GT359] (Abcam, catalog No. ab184607, 1:10,000) was used to detect His₆-tagged proteins, and acetyl lysine mouse monoclonal antibody (EasyBio, China, catalog No. BE3411, 1:2000) and Goat anti-mouse IgG (H&L) HRP-conjugated antibody (EasyBio, China, catalog No. BE0102, 1:10,000) was used for protein acetylation analysis. Then protein signal was detected using Immobilon Western HRP substrate (Millipore) and Fusion FX6 Imaging System (Vilber, France).

The concentrations of purified protein samples were determined by A280 absorption and fractionated on a 12% SDS-PAGE gel. Then, the protein samples were transferred to polyvinylidene difluoride (PVDF) membranes for 1.5 h at 15 V. The membrane was blocked at room temperature for 1 h using quick block western reagent (Beyotime, China). Acetyl lysine mouse monoclonal antibody (EasyBio, China, 1:2000) was used as the primary antibody and incubated overnight at 4°C, then Goat horseradish peroxidase-conjugated anti-mouse antibody diluted in PBST (EasyBio, China, 1:10,000) was used as the secondary antibody and incubated for 1 h. After washing three times with PBST, an enhanced chemiluminescence (ECL) system was used for signal detection according to the manufacturer’s instructions.