Western Blot Detection of TAP-tagged Proteins

Detection of TAP-tag protein by Western blot was performed as described in Ishikawa et al.^{37 (link)}. Yeast strains were aerobically cultured at 30 °C in 2 mL of YPD medium. Optical density at 660 nm (OD₆₆₀) was measured, and units of 1 OD₆₆₀ were harvested during the log phase. Cells were treated with 1 mL of 0.2 N NaOH for 5 min at room temperature and then suspended in 2× NuPAGE LDS sample buffer (Invitrogen) and heated to 70 °C for 10 min. Protein lysate in the supernatant was labeled with EzLabel FluoroNeo (ATTO) and subjected to polyacrylamide gel electrophoresis with lithium dodecyl sulfate (SDS-PAGE), followed by Western blot with PAP (Sigma-Aldrich) (1:2000) and peroxidase-conjugated secondary antibody (Nichirei Biosciences) (1:1000). We used a NuPAGE 4–12% Bis-Tris Gel (Invitrogen) for SDS-PAGE and an iBlot Transfer Stack PVDF membrane (Invitrogen) for Western blot. Chemiluminescence was induced by SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and detected on a LAS-4000 image analyzer (Fujifilm) using ImageQuant LAS 4000 (GE Healthcare).