Ab72569

For surface staining, HEK293FT cells were plated at 3×10 5 cells per well in 1 mL DMEM in 12-well plates and transfected as described above, using 200 μL transfection reagent per well. At 36-48 h after transfection, cells were harvested with 500 µL FACS buffer and spun at 150×g at 4°C for 5 min. For the experiment in which multiple harvest methods were compared (Supplementary Fig. 14), some samples were harvested using 0.05% Trypsin-EDTA (3 min or 10 min incubation, 37°C), which were then quenched with medium and added to two volumes of FACS buffer. Supernatant was decanted, and 50 µL fresh FACS buffer and 10 µL human IgG (Human IgG Isotype Control, ThermoFisher Scientific #02-7102, RRID: AB_2532958, stock concentration 1 mg/mL) was added. Cells were incubated in this mixture at 4°C for 5 min. 5 µL FLAG tag antibody (Anti-DDDDK-PE, Abcam ab72469, RRID: AB_1268475, or Anti-DDDDK-APC, Abcam ab72569, RRID: AB_1310127) was added at a concentration of 0.5 µg per sample and cells incubated at 4°C for 30 min. Following incubation, 1 mL FACS buffer was added, cells were spun at 150×g at 4°C for 5 min, and supernatant was decanted. This wash step was repeated two more times to total three washes. After decanting supernatant in the final wash, 1-3 drops of FACS buffer were added.