Coomassie brilliant blue protein analysis kit

Total cellular protein, carbohydrate and lipid contents were independently determined, and the cellular dry weight was measured. Algal cells were pelleted using centrifugation at 4,000 × g for 5 min, followed by rinsing in 1 mL of PBS (50 mM, pH 7.8). To determine the cellular protein content, the proteins were extracted according to a reported method (Zhang et al., 2013 (link)) and detected using a “Coomassie brilliant blue protein analysis kit” (Nanjing Jiancheng Bioengineering Institute, China) with bovine serum albumin as the standard. The carbohydrate content was determined using the phenol-sulfuric acid colorimetric method, with glucose as the standard (Li et al., 2014b (link)). The algal cells were concentrated at 4,500 × g for 10 min, vacuum dried and measured to determine the total dry weight, and the total lipids were then extracted using a mixture of chloroform and methanol (Lee et al., 2010 (link)).

Human peripheral blood lymphocyte separation solution (Tianjin Haoyang Biological Products Technology Co., Ltd.), Coomassie Brilliant Blue Protein Analysis Kit (Nanjing Jiancheng Bioengineering Research Institute), horseradish enzyme-labeled goat anti-rabbit IgG (imported packaging by Beijing Zhongshan Jinqiao Company), DAB coloring kit (imported packaging by Beijing Zhongshan Jinqiao Company), pre-stained protein molecular weight markers (American Thermo Scientific Company), PVDF membrane (Millipore Inc. USA), ECL enhanced chemiluminescence kit (Beijing TIANGEN Company), rabbit anti-actin polyclonal antibody (Proteintech, USA).

After treated with algicidal bacterium for 0, 6, 12, 24, and 48 h, algal cells were pelleted at 1160 × g for 5 min followed by washing in 1 mL of PBS (NaCl 8 g, KCl 0.2 g, Na₂HPO₄1.44 g, KH₂PO₄ 0.24 g, in 1 L distilled water, 50 mM, pH 7.8) twice, and then homogenized with an ultrasonic cell pulverizer (NingBo Scientiz Biotechnological Co., Ltd, China) 50 times at 80 W (ultrasonic time: 2 s; rest time: 3 s) at below 4°C. Then, the homogenate was centrifuged at 10,000 g for 10 min at 4°C. One milliliters supernatant was used to assay cellular protein which was detected using the “Coomassie brilliant blue protein analysis kit” (Nanjing Jiancheng Bioengineering Institute, China), using bovine serum albumin as the standard. The remaining supernatant were stored at −80°C until they were used to analyze the alteration of malondialdehyde (MDA, a byproduct of lipid peroxidation), and the activities of SOD, CAT, and POD. All the analysis methods followed the kit's operation manual, the samples were mixed with test liquid, and measured by microplate reader after heating with water bath.