Bca protein assay kit

Clinical PCa tissues and paired adjacent tissues were obtained, washed twice with 0.01 M PBS and lysed on ice with radioimmunoprecipitation assay (RIPA, Beijing Solarbio Science & Technology Co., Ltd.) a cell lysis reagent for 30 min. Tissues were centrifuged at 16,000 x g for 15 min at 4˚C to remove insoluble proteins. The concentration of extracted protein was determined by bicinchoninic acid (BCA) protein assay kit (Leagene, Inc.). A total of 50 µg of each total protein extract was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Bio-Rad Laboratories, Inc.). After being blocked at 23˚C for 1 h with 5% milk solution, membranes were washed three times with Tris-buffered saline-tween (0.1% Tween-20) solution. The membranes were then incubated with primary antibodies, rabbit anti-human SPC24 (Abcam; 1:1,000; cat. no. ab169786) and β-actin (Proteintech Group, Inc.; 1:1,000; cat. no. HRP-60008) at 4˚C overnight. After being washed with TBST, the membranes were incubated with horseradish peroxidase-labeled goat-anti-rabbit IgG secondary antibody (Abcam; 1:4,000; cat. no. ab6721) at 37˚C for 1 h. The membranes were processed for protein detection using the BeyoECL Plus Kit (Beyotime Institute of Biotechnology).

Protein adsorption onto the films was determined to examine whether nano coating would change the protein adsorption. The sample (6 mm in diameter) was immersed in phosphate-buffered saline (PBS) for 2 hours. The samples then were immersed in a bovine serum albumin (BSA (4.5 g/L); Sigma-Aldrich, St Louis, MO, USA) solution at 37°C for 12 hours. The disks then rinsed with fresh PBS, immersed in 1% sodium dodecyl sulfate (SDS)/PBS solution, and sonicated at room temperature for 20 minutes to completely detach the BSA from the scaffold.38 (link) A BCA Protein Assay Kit (Beijing Leagene Biotechnology Co., Ltd, China) was used to determine the amount of BSA adsorbed onto the sample (n=9).

The fruiting bodies were obtained in Changbai Mountain, Jilin Province, P.R.China. The 3-acetoxylanosta-8,24-dien-21-oic acid (FPOA) was extracted from the fruiting bodies of Fomitopsis pinicola. The fruiting bodies were extracted with ethanol and separated by silica gel column, elution with a mixture of petroleum ether and ethyl acetate to get FPOA. The purity of FPOA was >95%, as detected via HPLC. Antibodies against caspase-3, −9, Bcl-2, PARP and Bax were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). β-actin was obtained from Wuhan Sanying Biotechnology, (Wuhan, China). BCA protein assay kit was purchased from Beijing Leagene Biotech, Co., Ltd., (Beijing, China). An Annexin V-FITC Apoptosis Detection kit was obtained from Tianjin Sungene Biotech Co., Ltd., (Tianjin, China). MTT and all other reagents were obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).