Frac 950 collector

Thawed cells were disrupted by sonication and the protein purified by nickel affinity chromatography and concentrated NcCAR protein preparations were stored at −80 °C in 50 mM MES buffer, pH 7.5, containing 10 mM MgCl₂, 1 mM EDTA and 1 mM DTT. For determination of the kinetic parameters, the NcCAR preparation obtained from Ni‐affinity purification was further purified by gel filtration on an ÄKTAPurifier 100. A HighLoad^TM 16/60 SuperdexTM 200 pg column was used (GE Healthcare) with a flow rate of 1 mL min⁻¹ with 50 mM MES buffer, pH 7.5, containing 10 mM MgCl₂, 1 mM EDTA, and 1 mM DTT as the mobile phase. Fractions of 1 mL were collected in a Frac 950 collector (GE Healthcare) and the protein containing fractions were pooled and stored at −80 °C.