Bca protein assay kit

Agarose bound AAL and LCA were purchased from Vector Labs (Burlingame, CA). BCA protein assay kits were purchased from Bio-Rad(Hercules, CA).

After scarification, exposure of the trachea and clamping of the left main bronchus were done. A catheter was placed into the trachea, through which 8 ml sterile cold saline was instilled. The lavage fluid was gently rinsed in and out 3 times before collection. Recovered fluids were centrifuged at 400×g for 10 min at 4 ºC. The supernatants were separated in ice-cold tubes and stored at –80 ºC till used. Measurement of the total proteins in BAL was achieved by BCA protein assay kits (Bio-Rad, California, USA) at absorbance of 750 nm.

STZ, pioglitazone and glipizide were purchased from Sigma-Aldrich Chemical Co. (Skbio Life Sciences Technology, Beijing, China). Rat insulin ELISA kitS were purchased from Westang Biotechnology Co. (Shanghai, China). Glucose assay kits, cholesterol assay Kits, and triglyceride assay Kits were produced from BioSino Bio-technology and Science Co.(Beijing, China). Commercial fed a standard rodent chow (4% fat, 24% protein and 4.5% crude fiber), high-fat (15% fat) diet, high -cholesterol (0.5% cholesterol) diet, and high-fat and high-cholesterol (15% fat and 0.5% cholesterol) diet were purchased from Institute of Laboratory Animal Sciences , Chinese Academy of Medical Sciences. BCA protein assay kits were purchased from Bio-Rad Company (Bio-Rad Biotechnology, Beijing, China).

Each mouse was infected with 12 cercariae of S. japonicum. Six mice were randomly chosen from the infected and normal control groups at 0, 4, 7 and 11 weeks post-infection in each independent experiment, and all the mice were euthanized via diethyl ether-induced anesthesia for further study.
Schistosome egg antigen (SEA) was obtained from Jiangsu Institute of Parasitic Diseases, Wuxi, China, and prepared following a previously described method with some modifications [14 (link)]. Briefly, rabbits were infected by ~ 2000 cercariae, and their livers were collected and cut into a number of pieces six to seven weeks post-infection. Then, the liver pieces were homogenized in PBS on ice and then filtered, washed and centrifuged. Purified eggs in pre-cooled PBS were sonicated three times for 15 min. The suspension was frozen/thawed several times and centrifuged at 25,000×g for 30 min. A 0.22-μm filter was used to pass the supernatant and obtain SEA, which was evaluated for contaminating endotoxin (LPS) by the chromogenic LAL end-point assay kit (Cambrex, Charles City, IO, USA). The concentration of LPS in SEA was below 1.2 endotoxin units (EU)/mg. A bicinchoninic acid (BCA) Protein Assay kit (Bio-Rad, Richmond, CA, USA) was used to determine the protein concentration.

In each group, we randomly mixed five samples into one sample for the TMT proteome experiment. That is, three mixed bone samples in each group were prepared for further proteome analysis. Cancellous bone of the same mass at the same site of the right distal femur (3 mm to the rear and above where the growth plate disappears) from five rats was mixed into one sample (0.5 g) and ground with liquid nitrogen. 5 times volume of TCA/acetone (1:9) was added to the powder and mixed by vortex. The mixture was placed at −20°C for 4 h, and centrifuged at 6000 g for 40 min at 4°C. The supernatant was discarded. The pre-cooling acetone was added and washed for three times. The precipitation was air dried. 30 times volume of SDT buffer was added to 20–30 mg powder, mixed and boiled for 5 min. The lysate was sonicated and then boiled for 15 min. After centrifuged at 1,4000 g for 40 min, the supernatant was filtered with 0.22 µm filters. The filtrate was quantified with the BCA Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). The sample was stored at −80°C.

The renal tissues (30–40 mg) were lysed with RIPA buffer (25 mM Tris, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific, MA). Total protein concentration was measured using the BCA protein assay kit (Bio-Rad, CA, USA). After normalization, equal amounts of proteins were separated by 10% SDS–PAGE and transferred to PVDF membranes (0.45 μm, Millipore, Germany). The membranes were blocked with 5% nonfat milk in TBST for 2 h and incubated overnight with primary antibodies at 4°C. After three washes with TBST, the membranes were incubated with secondary antibodies (Thermo Fisher Scientific, 1:10,000) at room temperature for 1 h. Finally, the blots were washed with TBST three times and incubated using the EasySee western Blot Kit (Transgen Biotech, China) to visualize the immunoreactive bands.