Microson xl200 ultrasonic cell disruptor

Total protein was harvested from cells by standard methods. Briefly, cells were washed in PBS and trypsinized for 3 minutes at 37 degrees. Washed two more times in PBS and pellet collected each time. Pellets were sonicated in sonication medium (100mM Tris, 2mM EDTA, 250mM sucrose, pH 7.4) for 5 seconds at 60% power on a Misonix Microson XL200 Ultrasonic Cell Disruptor. Protein concentration was determined using Pierce BCA protein assay kit and samples adjusted to 1 mg/ul to perform spectrophotometric kinetic assays. Absorbances were read on a Synergy II microplate reader (Biotek). Enzymatic activity of Complexes I-IV and CS were measured as previously published (Kirby et al., 2007 (link)). Complex I activity was determined by measuring rotenone sensitive NADH oxidation (340 nm), complex II activity by measuring DCIP reduction (600 nm), complex III activity by measuring cytochrome c reduction (550 nm), complex IV activity by measuring oxidation of reduced cytochrome c. Citrate synthase activity by measuring the color of TNB (412 nm), which is generated from DTNB present in the reaction of citrate synthesis, and caused by the deacetylation of Acetyl-CoA. Activities were calculated as nmol/min/mg protein. All assays were conducted in triplicate and expressed as percentage of control.