Anti his tag mouse monoclonal antibody

T. reesei mycelia were harvested following a 24-h growth in glucose medium and ground with liquid nitrogen. The pulverized samples were then resuspended in Lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 0.02% NP-40 and 1 mM PMSF), followed by centrifugation at 12,000 rpm for 10 min at 4 °C to eliminate cell debris. The resulting supernatant was collected as intracellular proteins. The extracted protein samples were fractionated using SDS-PAGE and subsequently transferred to a cellulose acetate membrane. This membrane was then blocked with 10% skim milk in TBST and incubated with primary antibodies, including the anti His-tag mouse monoclonal antibody (Yeasen, Shanghai, China, #30401ES) at a dilution of 1:5000 and the anti-dimethyl arginine mouse monoclonal antibody (Abcam, #ab413) at a dilution of 1:500. Following to incubation with the primary antibodies, the membrane was washed three times with TBST and then incubated with Peroxidase AffiniPure Goat Anti-Mouse antibody (Yeasen, Shanghai, China, #33201ES). The membrane was washed three times in TBST prior to enhanced chemiluminescence detection (Tanon, Shanghai, China).

The recombinant GST and GST-ACE1^317-463aa were purified using Glutathione Beads (Smart-LifeSciences, China) from E. coli BL21 (DE3) following an 18-hour induction with 0.1 mM IPTG. Similarly, the recombinant His-TrSAM protein was purified using High Affinity Ni-NTA Resin (GenScript, Nanjing, China) from E.coli under identical condition. A weight of 20 μg purified GST or GST- ACE1^317-463aa fusion proteins was incubated with 20 μg His-TrSAM in 500 μL incubation buffer (50 mM Tris-HCl pH6.8, 250 mM NaCl, 1.5% glycerol, 0.6% Triton X-100 and 0.1% Tween-80) for 4 h at 4 °C. The beads were then washed three times with the incubation buffer. The washed beads were boiled in SDS loading buffer and subsequently separated followed by western blot analysis with anti-GST (Yeasen, Shanghai, China, #30901ES) and anti His-tag mouse monoclonal antibody (Yeasen, Shanghai, China, #30401ES).