Coomassie brilliant blue assay kit

The content of protein was determined by Coomassie brilliant blue assay kit (Nanjing Jiancheng, China). In detail, 10 mL of C. cryptica culture was centrifuged (3500 rpm, 15 min) at room temperature. The supernatant was discarded and the harvested cell pellet was resuspended in 5 mL of double distilled water (dd-water) and was crushed by an ultrasonic cell crusher under 25% power for 20 min (Ningbo Scientz Biotechnology CO..LTD). The concentration of soluble protein in the supernatant was measured by the assay kit.

The levels of oxidative stress in kidney were quantified by measuring malondialdehyde (MDA) using the MDA activity assay kit; MDA is one of the final products of polyunsaturated fatty acid peroxidation. The free radicals cause overproduction of MDA; therefore, the level of MDA is used as a marker of oxidative stress and lipid peroxidation (Gaweł et al., 2004 (link)). Superoxide dismutase (SOD) activity was measured using an SOD activity assay kit (Beyotime Biotechnology). In brief, renal tissues were homogenized using a homogenizer (Jingxin Experimental Technology, Shanghai, China); the homogenate supernatant was collected by centrifuging at 12,000g for 15 min at 4°C, which was used for measuring MDA at an absorbance 530–540 nm and SOD at 560 nm. The levels of MDA and SOD were standardization to unit weight of total protein content.
On the last day of the 12th week of the experiment, the urine was collected by using metabolic cage and centrifuged at 4,000 revolutions/min for 15 min at 4°C to obtain urine supernatant. Coomassie brilliant blue assay kit (Jiancheng Bioengineering Institute, Nanjing, China) was used to quantify the levels of urine albumin.