Peroxidase affinipure goat anti mouse 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 proteins TrSAM and ACE1^317-463aa were individually fused with the GST protein tag. Subsequently, these fusion proteins were introduced into the expression host E. Coli BL21 (DE3), followed by an 18-hour induction using 0.1 mM IPTG. The recombinant GST-TrSAM and GST-ACE1^317-463aa proteins were purified using Glutathione Beads (Smart-LifeSciences, China) and employed as the methyltransferase (5 μg) and substrate (10 μg) respectively for methylation. S-AdoMet served as the methyl donor. The methylation solutions were incubated in reaction buffer (50 mM Tris-HCl pH 8.5, 20 mM KCl, 10 mM MgCl₂, 100 mM sucrose and 1 mM β-mercaptoethanol) at 28 °C for specific durations and subsequently fractionated by SDS-PAGE. Western blot analysis was performed using the anti-dimethyl arginine mouse monoclonal antibody (Abcam, #ab413) and Peroxidase AffiniPure Goat Anti-Mouse antibody (Yeasen, Shanghai, China, #33201ES).