Bradford protein quantification kit

A single E. coli BL21 (DE3) colony was transferred into 5 ml LB medium containing 50 μg/ml kanamycin, and the culture was shaken at 37 °C for 16 h. The cells were then collected, resuspended in 400 ml LB medium, and shaken at 200 rpm and 37 °C until the A₆₀₀ value reached 0.5. Recombinant protein expression was induced by adding 1 mM IPTG (400 μl) to the fermentation broth. After 4 h of shaking at 25 °C, cells were harvested by centrifugation at 5000g for 20 min, resuspended in lysis buffer (50 mM Tris, 100 mM NaCl, and 1% (v/v) Triton X-100; pH adjusted to 7.4), and disrupted by sonication (40 ON/OFF cycles with 20 μm probe amplitude for 15 s) using an ultrasonic homogenizer (JY92-IN, Ningbo Scientz Biotechnology).
The cell lysate supernatant was collected by centrifugation at 10,000g for 30 min and loaded onto a Ni-NTA affinity column (Wuxi Tianyan). After washing the column with 50 ml of washing buffer (50 mM Tris/HCl; pH adjusted to 7.2), the recombinant enzymes were then eluted with elution buffer (50 mM Tris/HCl and 300 mM imidazole; pH adjusted to 7.4). Protein expression and purification were monitored by SDS-PAGE using Coomassie brilliant blue G-250. Elution fractions showing the highest purity were pooled and stored in 20% glycerol (v/v) at −80 °C. The protein concentration was measured using a Bradford Protein Quantification Kit (Sangon Biotech).

The crude enzyme was prepared according to the method reported by Trigoso et al. [36 (link)]. After centrifugation at 4 °C for 30 min at 10,000×g, the cell-free supernatants were immediately used to determine the enzyme activities. Protein concentrations were determined using the Bradford Protein Quantification Kit (Sangon, Shanghai, China) with bovine serum albumin as standard. The analyses of enzyme activities and protein concentrations were done in triplicate. Specific activity was given as the number of U mg⁻¹ of protein. The analyses of enzymes GAPDH and IDH were based on the protocol of Xu et al. [17 (link)] and Wang et al. [37 (link)], respectively.