Western blot analysis of c-Myc-chPylRS-6xHis variants

Cell lysates (30 μL) of expressed protein were combined with 25 μL of XT Sample Buffer (Bio-Rad), 5 μL of 2-mercaptoethanol, and 40 μL water. The samples were heated at 70°C for 10 min and 7.5 μL of prepared sample was loaded per well of a Bolt Bis-Tris Plus Gel (Thermo Fisher Scientific). Precision Plus Protein Dual Color Standard (4 μL) Bio-Rad was used as the reference ladder. The loaded gel was run at 200V for 22 min in 1x Bolt MES SDS running buffer (Thermo Fisher Scientific). The gel was transferred to a PVDF membrane using the iBlot 2 Gel Transfer Device (Thermo Fisher Scientific). The membrane was blocked for 1 h at room temperature in 50% Odyssey blocking buffer (PBS) (Li-Cor) and was then soaked 4 times for 5 min in PBS containing 0.1% Tween-20 (PBST). The blocked membrane was soaked with primary antibodies [rabbit anti-6xHis (1:1,000 dilution) (Abcam, ab9108) and mouse anti-c-Myc (1:7,000 dilution) (Sigma-Aldrich, M4439)] in 50% Odyssey buffer (PBS) containing 0.2% Tween-20 for 4 h at room temperature. The membrane was washed 4 times in PBST, and then soaked for 1 h in the dark at room temperature with secondary antibodies [donkey anti-mouse 800CW (1:20,000 dilution) (Li-Cor) and goat anti-rabbit 680RD (1:20,000 dilution) (Li-Cor)] in Odyssey buffer containing 0.01% SDS, 0.2% Tween-20. The membrane was washed 4 times in PBST and finally rinsed with PBS. The membrane was scanned using an Odyssey Imaging System (Li-Cor).