Bca assay kit

The total protein from GC cells were extracted using radioimmune precipitation (RIPA) buffer (Wolsen Biotech,) supplemented with phosphatase and protease inhibitors as well as phenylmethyl sulfonyl (PMSF) (Sigma). A BCA assay kit (Takara Bio, Inc.) was then used for quantifying protein concentration. After that, prepared samples were isolated via SDS‐PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, CA). Ten per cent skim milk was then used for blocking for 90 min, followed by incubating overnight at 4°C using appropriate primary antibodies. The membranes were treated with HRP‐linked secondary antibodies for 1.5 h on the second day (Cell Signalling Technology, Inc.). The membranes were cleaned 3 times with Tris‐buffered saline‐Tween 20 after each incubation step. At the end, the membranes were observed using an enhanced chemiluminescence detection system (Syngene Europe) and analysed with ImageJ software (National Institutes of Health). For primary antibodies used in this study, see Table 3.

Proteins were extracted from HCN cells and the protein concentrations were assessed using a BCA assay kit (TaKaRa BIO INC, Japan). The protein samples were resolved in a 10-12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Proteins were then transferred to polyvinylidene fluoride (PVDF) membrane, and blocked with 5% nonfat milk in Trisbuffered saline-Tween (TBST) 20 for 2 h at room temperature. Membranes were then incubated with primary antibody overnight. The antibodies were shown as follows: anti-Caspase-3, anti-Bcl2, anti-IL1, anti-IL6 and anti-NF-kb (1:1000; Santa Cruz Biotechnology,USA). An anti-GAPDH antibody was used as a loading control. Membranes were washed and incubated for 2 h in the presence of appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. The positive reaction was visualized by using 3, 3’-diaminobenzitine (DAB) solution (Sigma, St. Louis, MO) with a chemiluminescent Immobilon Western blotting detection system.

Cells were washed twice with cold PBS and then were lysed in the radio immunoprecipitation assay buffer (Bocai Biotechnology, Shanghai, China) supplemented with a protease inhibitor (Amyjet Scientific Inc, Wuhan, China). Lysate was centrifuged at 12 000 g for 15 minutes at 4°C. The protein concentrations in the supernatants were measured using the BCA assay kit (Takara, Japan) as the manufacturer's instructions. Sample proteins (50 μg) were subjected to 10% polyacrylamide gel containing sodium dodecyl and then transferred into the polyvinylidene fluoride membrane. After being blocked with 5% free‐fat milk in Tween 20‐containing Tris‐buffered saline for 2 hours at 4°C, the membranes were incubated with primary antibodies over night at 4°C (listed in Table 1). Then, membranes were washed with Tween 20‐containing Tris‐buffered saline for five times and incubated with horseradish peroxidase‐conjugated secondary antibody (1:5000, Bioword, MN, USA) for 1 hour at room temperature and then were washed with the buffer for three times again. The protein bands were visualized with enhanced chemiluminescence (Pierce Chemical Co, IL, USA). The intensities of proteins were quantified using Image J software.

Cell lysates were prepared with the “RIPA” buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate) supplemented with phenylmethylsulfonyl fluoride and Na₃VO₄ at 1 mM. Samples were subjected to centrifugation clearance before protein concentration determination with a BCA assay kit (Takara). Samples containing equal protein amounts were mixed with loading buffer and denatured for 5 min at 95 °C. After loading onto SDS-polyacrylamide gels, proteins were separated and then transferred to nitrocellulose membranes, which were blocked with 4% fat-free milk and incubated with primary antibodies at 4 °C overnight. Following PBS washes, the membrane was incubated with secondary antibodies for 1 h at room temperature. Finally, membranes were scanned on a LI-COR Odyssey imager, and protein band intensities were analyzed with the Image Studio programme (version 4.0).

Amino acid concentration was tested using ninhydrin reagent^{45 (link)}. FDB culture broth was precipitated using 20% TCA and 200 μL of supernatant mixed with 50 μL phosphate buffer (pH 8.04) and 50 μL 2% (w v⁻¹) ninhydrin reagent. The mixture was heated in water bath at 90 °C for 30 min, followed by addition of 950 μL distilled water. Absorbance was read at 570 nm to quantify the amino acids present in the hydrolysate according to a prepared isoleucine standard curve. Amino acid composition and content were further evaluated and quantified using an Amino acid analyzer A-300 advanced (MembraPure, Germany). A bicinchoninic acid (BCA) assay kit from TaKaRa (Shanghai, China) was used to determine the soluble protein concentrations in fermentation solutions, using bovine serum albumin as the standard. Each sample was assessed in triplicate.
The release of sulfhydryl groups into the FDB culture medium was determined spectrophotometrically, according to the method of Ellman^{46 (link)}. DTNB (10 μL) and 0.1 M phosphate buffer (500 μL; 1 mM EDTA, pH 8.0) was added to 50 μL of extracellular broth mixture. Absorbance was measured at 420 nm and the concentration of sulfhydryl groups calculated.

Cultured cells were lysed in 2× SDS-containing buffer, and the protein concentrations were quantified using BCA assay kit (Takara Bio). Protein samples were then reduced and boiled. Subsequently, equal amounts of protein were applied into each lane and separated in an 8% polyacrylamide tris glycine SDS gel, then transferred to a nitrocellulose membrane. The membranes were blocked in 2% skim milk-containing TBS supplemented with 0.1% Tween-20 for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. Following a washing step, the membranes were incubated with HRP-conjugated secondary antibodies. Signals were developed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific), and acquired with Image Quant LAS 4000 mini (GE Healthcare). Antibodies used in this study were rabbit polyclonal anti-Ror2 antibodies (BD Biosciences), mouse monoclonal anti-β-actin antibodies (Sigma-Aldrich), anti-rabbit IgG-HRP goat antibodies (Jackson), and anti-mouse IgG-HRP antibodies (Jackson).