Quantipro bicinchoninic acid bca assay kit

A/California/07/09 (H1N1) (kindly provided by Korea Centers for Disease Control and Prevention) was propagated in the allantoic cavities of 9–11 day old specific pathogen free (SPF) embryonated chicken eggs (ECE). Seventy-two hours after inoculation at 37°C, allantoic fluid containing H1N1 virus was collected and clarified by low speed centrifugation (2,000 × g, 30 min, 4°C), and chemically treated with formalin (final concentration of 0.2%) for 24 h at 22°C for virus inactivation. Formalin-treated allantoic fluid was stored at 4°C until the confirmation of virus inactivation. The inactivation of virus was confirmed by the inoculation of 0.2 ml of formalin-treated allantoic fluid into five 10 days-old-embryonic eggs. After 72 h of incubation at 37°C, the allantoic fluids from all ECEs showed negative results for hemagglutination activity with chicken red blood cell (RBC). After the confirmation of inactivation, the inactivated H1N1 virus from the clarified supernatants was pelleted (30,000 × g, 1.5 h, 4°C). The pelleted virus was resuspended in phosphate-buffered saline (PBS) solution (pH 7.4) and purified using 20–50% (w/v) discontinuous sucrose density gradient purification (150,000 × g, 2.5 h, 4°C). The protein concentration of purified inactivated virus was determined by QuantiPro Bicinchoninic Acid (BCA) Assay kit (Sigma) according to the manufacturer's instructions.

Coptidis Rhizoma (Coptis chinensis Franch.) was purchased from Shanghai Kang Qiao Herbal Pieces Co., Ltd. (China), which is a GMP-certificated manufacturer. The herb was authenticated by Prof. Zhi-Li Zhao of the Department of Botany, Shanghai University of Traditional Chinese Medicine according to The Pharmacopoeia of People’s Republic of China (2010 edition). All Coptidis Rhizoma alkaloid standards were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Carbamazepine and the QuantiPro bicinchoninic acid (BCA) assay kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile was purchased from Merck (Darmstadt, Germany). Tissue-Tek O.C.T. Compound was obtained from Sakura Finetek (Torrance, CA, USA). The pure water used in the current study was prepared using a Milli-Q system (Millipore Corporation, Billerica, MA, USA). All other materials were of analytical grade or higher.