Amicon ultra 10 k centrifugal filter device

Plasmid harboring METTL4 was transfected into HEK293T cells following the manufacturer’s protocol. Cells were grown at 37 °C with 5% CO₂ and harvested after 48 hr. To purify METTL4 protein, cells were suspended in 2 volumes of lysis buffer (150 mM KCl, 10 mM HEPES (pH 7.6), 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, protease inhibitor cocktail) followed by rotating at 4 °C for 30 min to lysis the cell completely. The lysate solution was centrifuged at 13000 rpm for 30 min at 4 °C. The resultant supernatant was passed through a 0.22 μm membrane syringe filter and then incubated with anti-Flag M2 magnetic beads (Sigma-Aldrich) at 4 °C on a rotate wheel for 3 hr. Afterwards, the protein-beads complex was washed with ice-cold B&W buffer (200 mM NaCl, 50 mM HEPES (pH 7.6), 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, protease inhibitor cocktail) and METTL4 protein was eluted twice by incubation with 300 μl elution buffer (200 mM KCl, 50 mM Tris-HCl pH 8.0, 1.5 mM MgCl₂, 0.5 mM DTT, 1× proteinase inhibitor cocktail, 0.5 mg/ml 3× Flag peptide (Genscript)) for 30 min at 4 °C. The protein was concentrated by using Amicon Ultra 10K centrifugal filter Device (Millipore).