Dylight 488 550 maleimide

Antibodies in the hybridoma culture supernatants were collected using Protein A Sepharose CL-4B beads (GE Healthcare) at 4°C overnight. The beads were suspended in a 5-fold volume of PBS (pH 7.0) containing 10 mM ethylenediaminetetraacetic acid (PBS-EDTA; pH 7.0). DyLight 488/550 Maleimide (Thermo Fisher Scientific) was used to label antibodies via free Cysteine residues (Huh et al., 2013 (link)), which was dissolved in dimethyl sulfoxide at 10 μg/μL immediately before use and added to the bead suspension at 0.5 mM. Dye-to-protein molecular ratio was maintained at approximately 1:30 including the Protein A on the beads. The reaction was performed on a tube rotator for 2 h at RT, and unbound dye was carefully and repeatedly washed away using PBS and centrifuged at 500 × g for 2 min. The beads were incubated in digestion buffer (50 mM Tris-HCl, 10 mM cysteine-HCl, 2 mM EDTA, pH 8.0) containing 0.01 mg/mL papain (nacalai tesque, Inc) for 1 h at 37° C water bath. Cleaved fluorescently-labeled Fab fragments (Fab probes) were recovered by centrifugation at 500 × g for 2 min and supplemented by 1 μg/mL of leupeptin (Peptide Institute).