Novaspec plus

Rovalpituzumab (1 mg, 6.8 nmol) was incubated with IR700 NHS ester (LI-COR Biosciences, Lincoln, NE, USA) (66.8 mg, 34.2 nmol, 5 mmol/L in DMSO) in 0.1 mol/L Na₂HPO₄ (pH 8.6) at room temperature for 1 h. The mixture was purified using a Sephadex G50 column (PD-10, GE Healthcare; Piscataway, NJ, USA). The protein concentration was determined using a Coomassie Plus protein assay kit (Thermo Fisher Scientific) by spectroscopically measuring the absorption at 595 nm (Novaspec Plus; GE Healthcare). The concentration of IR700 was measured via absorption at 689 nm using spectroscopy to confirm the number of mAb-conjugated fluorophores. The synthesis was controlled so that an average of 3 IR700 molecules were bound to a single antibody; these rovalpituzumab conjugates are referred to as rova-IR700.

Anti-GPR87 antibody (GPR87ab) (1 mg; 6.8 nmol) was incubated with IR700 NHS ester (LI-COR Biosciences, Lincoln, NE, USA) (66.8 mg; 34.2 nmol, 5 mmol/L in DMSO) in 0.1 mol/L Na₂HPO₄ (pH 8.6) at 15–25 °C for 1 h. The mixture was purified employing a Sephadex G50 column (PD-10, GE Healthcare; Piscataway, NJ, USA; cat # 17,085,101). The protein concentration was decided employing a Coomassie Plus protein assay kit (Thermo Fisher Scientific; cat # 23,236) by spectroscopically (at 595 nm) (Novaspec Plus; GE Healthcare; cat # 10,187,054). The concentration of IR700 was spectroscopically measured at 689 nm to verify the amount of Ab-conjugated fluorophores [23] (link), [24] (link), [25] (link). The synthesis was controlled in order that a mean of three IR700 molecules were sure to a single antibody; these GPR87ab conjugates with IR700 were abbreviated to as GPR87ab-IR700.