Dye-conjugated Fab Preparation Protocol

Dye-conjugated Fabs were prepared as previously described (Kimura and Yamagata, 2015 ). In brief, 100 μg purified Fab (anti-H3K9ac: CMA310/19E5, Hayashi-Takanaka et al., 2011 (link); anti-H3S28ph: CMA315/10-20F11, Hayashi-Takanaka et al., 2014 (link); anti-DYKDDDDK tag: Fujifilm Wako, 012-22384) was reacted with N-hydroxysuccinimide ester-conjugated fluorescence dye (Cy5, cytiva, PF11A25001; Cy3, cytiva, PA13105) in 100 mM NaHCO₃ (pH 8.3) in PBS for 1 h at room temperature in the dark. Dye-conjugated Fabs were collected using PD MiniTrap G-25 column (Cytiva, 28918004; pre-equilibrated with PBS) and were concentrated to 1 mg/ml using a 10-kDa cutoff Amicon Ultracell Centrifuge Filter Unit (Merck, UFC5010BK), and stored at 4°C in the dark.