Cut off dialysis cup

Goat anti-mouse IgG (100 µL, Jackson ImmunoResearch, product number 115-005-146 (Fig. 4c) or Thermo Fisher Scientific, product number 31160 (Fig. 6a and Supplementary Fig. 12)) was dialyzed against conjugation buffer (150 mM NaCl and 10 mM HEPES, pH 8) at room temperature and 4 °C (3 × 300 mL; 2 hours, 2 hours, and overnight), using a 10-kDa molecular weight cut-off dialysis cup (Thermo Scientific). A ~ 1 mM solution of Eu³⁺/DTBTA or Alexa Fluor 594 NHS ester (Thermo Fisher Scientific, product number A20004) in ice-cold conjugation buffer was prepared immediately before use, and 5 µL of this solution was added to 100 µL of the dialyzed antibody solution. After the reaction mixture was incubated at room temperature overnight, unreacted Eu³⁺/DTBTA was removed by two rounds of size-exclusion chromatography (Illustra MicroSpin G-50; GE Healthcare Life Sciences). The resulting stock solution of Eu³⁺/DTBTA-labeled and Alexa Fluor 594-labeled secondary antibody appeared red-fluorescent upon 365-nm illumination. The probe-to-antibody ratios were calculated from absorbance levels at 341 nm (Eu³⁺/DTBTA), 594 nm (Alexa Fluor 594), and 280 nm (antibody) as determined with a NanoDrop spectrophotometer (Thermo Scientific).