Hitrap columns for ni nta affinity chromatography

Reagents used for molecular biology experiments were purchased from New England BioLabs (Ipswich, MA), Thermo Fisher Scientific (Waltham, MA), or Gold Biotechnology Inc. (St. Louis, MO). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Escherichia coli DH5α and BL21 (DE3) strains were used for plasmid maintenance and protein overexpression, respectively. Co-expression vector pRSFDuet-1 was obtained from Novagen. MALDI-TOF-MS analysis was performed using a Bruker UltrafleXtreme MALDI TOF-TOF mass spectrometer (Bruker Daltonics) in reflector positive mode at the University of Illinois School of Chemical Sciences Mass Spectrometry Laboratory. ESI-MS/MS analyses were performed using a SYNAPT ESI quadrupole TOF Mass Spectrometry System (Waters) equipped with an ACQUITY Ultra Performance Liquid Chromatography (UPLC) system (Waters). ³¹P NMR spectra were recorded on an Agilent 600 MHz spectrometer in 10% D₂O. HiTrap columns for Ni-NTA affinity chromatography were purchased from GE Healthcare.