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Human cathepsin B-mediated cleavage assay

CY Chisato M. Yamazaki
AY Aiko Yamaguchi
YA Yasuaki Anami
WX Wei Xiong
YO Yoshihiro Otani
JL Jangsoon Lee
NU Naoto T. Ueno
NZ Ningyan Zhang
ZA Zhiqiang An
KT Kyoji Tsuchikama
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Each ADC (1 mg mL−1) in 30 µL of MES buffer (10 mM MES-Na, 40 µM dithiothreitol pH 5.0) was incubated at 37 °C for 10 min. To the solution was added pre-warmed human cathepsin B (20 ng µL−1, EMD Millipore) in 30 µL MES buffer, followed by incubation at 37 °C. Aliquots (20 µL) were collected at each time point (4, 8, and 24 h) and treated with EDTA-free protease inhibitor cocktails (0.5 µL of 100× solution, Thermo Scientific). All samples were analyzed using an Agilent 1100 HPLC system equipped with a MabPac RP column (3 × 50 mm, 4 µm, Thermo Scientific). Elution conditions were as follows: Mobile phase A = water (0.1% formic acid); mobile phase B = acetonitrile (0.1% formic acid); gradient over 6.8 min from A : B = 75 : 25 to 1 : 99; flow rate = 0.5 mL min−1. Average DAR values were determined based on UV peak areas.

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