Kinase Inhibitor Sepharose Conjugation

Kinase inhibitors were conjugated with local laboratory generated ECH Sepharose 4B using the carbodiimide coupling method (12, 13 (link)). Briefly, nine kinase inhibitors (Palbociclib, Crizotinib, GSK690693, AZD4547, CZC-8004, Afatinib, FRAX597, Abemaciclib, and Axitinib; Supplementary Table S1) were separately conjugated to homemade ECH Sepharose 4B via carbodiimide coupling chemistry as described previously (12 (link)). ECH Sepharose 4B was synthesized using conjugating 6-Aminohexanoic acid (Sigma) to cyanogen bromide (CNBr)-activated Sepharose 4B (GE Healthcare). Each kinase inhibitor was dissolved in 50% dimethylformamide (DMF)/ethanol (EtOH) and added to the ECH Sepharose 4B beads in the presence of 0.1 mol/L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and allowed to react overnight at 4°C with rotation. After coupling, the unreacted groups were inactivated with ethanolamine. Subsequently, beads were washed with 0.1 mol/L Tris-HCl, pH 8.3 with 500 mmol/L NaCl and 0.1 mol/L acetate, pH 4.0 with 500 mmol/L NaCl and stored in 20% ethanol at 4°C in the dark.