GO-PEG-NH2 was mixed with S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) or FITC, which has the same chemical reaction between the SCN group and the NH2 group on GO, at a molar ratio of 1:10 at pH 9.0 for 2 h. The resulting NOTA-GO (or FITC-GO) was purified by centrifugation filtration using 100 kDa cutoff Amicon filters (15 minutes at 12,520 g). NOTA-GO (or FITC-GO) was subsequently reacted with succinimidyl carboxymethyl PEG maleimide (SCM-PEG-Mal) at pH 8.5 at a molar ratio of 1:30 for 2 h. After removing the unreacted SCM-PEG-Mal and other reagents by centrifugation filtration, the resulting reaction intermediates were named as NOTA-GO-Mal or FITC-GO-Mal.
Meanwhile, TRC105 was mixed with Traut’s reagent at a molar ratio of 1:25 at pH 8.0. After 2 h of incubation at room temperature (RT), the resulting TRC105-SH was purified by size exclusive chromatography using phosphate-buffered saline (PBS, pre-treated with Chelex 100 resin to prevent oxidation of the thiol) as the mobile phase. With this reaction ratio, we calculated that there are about 8 thiol groups per TRC105 molecule based on Ellman’s reagent titration (seeSupporting Information Figure S2 ). Subsequently, NOTA-GO-Mal or FITC-GO-Mal was mixed with TRC105-SH at a molar ratio of 1:5 at pH 7.5 in the presence of tris(2-carboxyethyl)phosphine (i.e. TCEP) to avoid disulfide formation between TRC105-SH. The final products were purified by centrifugation filtration as described above and termed NOTA-GO-TRC105 and FITC-GO-TRC105.
Meanwhile, TRC105 was mixed with Traut’s reagent at a molar ratio of 1:25 at pH 8.0. After 2 h of incubation at room temperature (RT), the resulting TRC105-SH was purified by size exclusive chromatography using phosphate-buffered saline (PBS, pre-treated with Chelex 100 resin to prevent oxidation of the thiol) as the mobile phase. With this reaction ratio, we calculated that there are about 8 thiol groups per TRC105 molecule based on Ellman’s reagent titration (see
