1. Linker: Erbitux was reacted with dibenzocyclooctyne-(polyethylene glycol) 4-N-hydroxysuccinimidyl ester (DBCO-PEG4-NHS) cross-linker.

2. Reaction buffer: All of the reaction was carried out in 1 × PBS. The optimal ratio of Erbitux/dye/linker is 1:10:20.

3. First step: For a typical reaction, 300 μl of Erbitux (13.7 μM) and 26.7 μl of DBCO-PEG4-NHS (3.08 mM in DMSO) were mixed. The mixture was vortexed, followed by incubation at a shaker or stirring through magnetic beads for 2 hours.

4. Washing: After the reaction, the product was washed by the Amicon Centrifugal Filter (30 kDa) four times against a PBS buffer. The final volume is 300 μl.

5. Second step: Add 101 μl of IR-FGP (408.5 μM) to 100 μl of Erbitux-DBCO (the concentration of the dye can be tuned). The mixture was vortexed, followed by incubation at a shaker or stirring through magnetic beads for 3 hours.

6. The product (the output can be enlarged on the basis of the same ratio) was subjected to DGU purification. Sucrose column gradient for 4 ml of DGU tube was made by interval adding (10, 15, 20, 25, 30, 35, and 40%; 400 μl of each), tilt-permeated, and then subjected to ultracentrifuge for 18 hours at 50,000 rpm at 4°C.

7. The DGU sample was collected by fractionating.

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