Coomassie brilliant blue protein assay kit

Approximately 0.4 g minced breast muscle of each sample was homogenized (w/v, 1/4) in ice-cold 154 mmol/L sterile sodium chloride solution using a homogenizer (PRO-PK-02200D, Pro Scientific, Inc., Monroe, CT, USA). Homogenate was thereafter centrifuged at 4450× g for 15 min at 4 °C, and the supernatant was stored at −80 °C for further analysis. Concentrations of malondiadehyde (MDA) and reduced glutathione (GSH) and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) were determined according to the manufacturer’s instructions using available commercial kits (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China). Total protein concentration of breast muscle was measured by a Coomassie brilliant blue protein assay kit purchased from Nanjing Jiancheng Institute of Bioengineering (Nanjing, China). The results were normalized against total protein concentration in each sample for inter-sample comparison.

Compound Danshen Tablet (CDT) was produced by Hutchison Whampoa Guangzhou Baiyunshan Chinese Medicine Co, Ltd (batch number: Z44023372, Guangzhou, China). The formula consisted of Savia miltiorrhiza, Panax Notoginseng, Borneol in proportions of 450:141:8. The main active ingredient were identified as tanshinone, cryptotanshinone, dihydrotanshinone I, tanshinone IIA, salvianolic acid B, sodium danshensu, rosmarinic acid and lithospermic acid by HPLC fingerprint [18 ]. The tablets were grinded and dissolved in sterile saline before use.
Huperzine A (Purity ≥ 99%, HPLC), acetylcholinesterase inhibitor, was produced by Chengdu Must Biotechnology Co, Ltd (batch number: MusT-11041101, Chengdu, China) and also dissolved in sterile saline before use. Coomassie brilliant blue protein assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Amyloid β-protein fragment 25–35 (Aβ_25-35) was purchased from Beijing Biosynthesis Biotechnology Co, LTD (Beijing, China).

The glycogen content of the muscle was measured according to the method previously described59 (link). Briefly, powdered muscle (50 mg) was hydrolyzed in 150 μl of 1 mol/L KOH by heating at 95 °C for 20 min. The samples were incubated at 37 °C for 2 h, centrifuged at 4 °C/16000 × g for 10 min, and neutralized with NaOH. The resulting free glycosyl units were determined using D-glucose as a standard, and the results are expressed as mg/g muscle tissue. The LPL activity and TG content in the skeletal muscle were measured using a Triglyceride Assay Kit and a Lipoprotein Lipase Assay Kit provided by the Nanjing Jiancheng Bioengineering Institute, China. Approximately 100 mg of frozen muscle was minced, weighed and thoroughly homogenized with 1 ml of ice-cold PBS using a mechanical tissue disrupter. After centrifugation (2500 × g at 4 °C), the supernatants were decanted and saved for assaying LPL activity, and the TG concentration was measured in triplicate at the appropriate dilutions. The LPL activity is expressed as U/mg protein of muscle tissue, and the TG content is expressed as mmol/g protein of muscle tissue. The total protein content of the supernatant was measured with the Coomassie Brilliant Blue Protein Assay Kit (Nanjing Jiancheng Institute of Bioengineering, China).

Throughout the experiment, all rats were placed in metabolic cages with adequate standard laboratory food and tap water, to collect 24-hour urine on a weekly basis. The 24-hour urine total protein concentration of collected urine was then analyzed with the Coomassie brilliant blue protein assay kit [21 (link)] (Nanjing Jiancheng Bioengineering Institute, China) and measured with an ultraviolet spectrophotometer (UV-2600, Shimadzu, Tokyo, Japan).