Microson ultrasonic cell disruptor xl2000

A total of 1 × 10⁶ cells were resuspended in 200 µL of mannitol buffer (10 mM Tris-HCl pH 7.2; 225 mM mannitol; 75 mM sucrose; 0.1 mM EDTA) and homogenized by sonication for 5 s at 60% intensity with Microson Ultrasonic Cell Disruptor XL2000 (Misonix). Then, the homogenates were centrifuged at 650 × g for 20 min at 4 °C, and the protein concentration of the resulting supernatants was quantified by Bradford assay. Homogenates were brought to 1 mg/ml in mannitol buffer. NADH oxidation was determined after electron transfer to ubiquinone by monitoring the decrease in 340 nm absorbance^{64 (link),65 (link)}. Briefly, 20 µg of protein homogenate were mixed with 100 µM decilubiquinone, 3.75 mg/mL BSA, and 50 mM K₂HPO₄ pH 7.5 in the presence or absence of 12.5 µM of rotenone. The reaction was initiated by adding 100 µM NADH at 37 °C, and light absorbance was monitored at 340 nm for 3 min in a UV-2401PC UV-VIS recording spectrophotometer (Shimadzu Corporation).

The E. coli BL21(DE3) and Origami™ B(DE3) strains transformed with the plasmid pET-16b-HLA-Crps were inoculated into 5 mL of LB medium containing 50 µg/mL ampicillin and incubated at 37 °C with shaking at 180 rpm until the absorbance at 600 nm reached 1.0. Expression was induced by the addition of IPTG, and the bacterial liquid was subjected to shaking at 180 rpm for 4 h. The cells were collected by centrifugation at 10,000 rpm for 3 min and washed with disruption buffer (20 mM Tris-HCl (pH 8) and 1 mM EDTA). One milliliter of the medium was resuspended in 100 µL of buffer and disrupted with a sonicator (Misonix™ Microson™ Ultrasonic Cell Disruptor XL2000) on ice. The mixture was centrifuged at 10,000 rpm for 3 min. The amount of protein in the obtained supernatant and pellet was confirmed by Tricine-SDS-PAGE (n = 3 for each).