Bca protein quantification kit

Western blot analysis was conducted to analyze protein expression in the lung tissue. Briefly, protein lysis buffer was added to minced lung tissue and the tissue was homogenized. The samples were centrifuged at 10,000 g at 4 °C. Protein concentration in the supernatant was quantified using a BCA protein quantification kit (Tiangen, Beijing, China). Aliquots containing 50 μg total proteins were used for 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the resolved proteins were transferred to a nitrocellulose membrane (BioTrace Medical, Menlo Park, CA, USA). The membranes were incubated with 1:1000 dilutions of antibodies against pulmonary surfactant protein (SP)-A, SP-C, SP-D, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (all from Cell Signaling Technology, Danvers, MA, USA) at 4 °C on a rotating shaker overnight. The membranes were subsequently washed with Tris buffered saline-Tween (TBST) three times for 5 min each time, followed by incubation with horseradish peroxidase-labeled goat anti-rabbit IgG (H + L; Beijing ZSGB Bio, Beijing, China, 1:5000 dilution) for 1 h at 25 °C on a horizontal shaker. The membranes were washed with TBST twice for 5 min each time, followed by chemiluminescence detection. The protein band areas were quantified using Image Lab 6.0 software (Bio-Rad, Hercules, CA, USA). The GAPDH antibody was used as an internal reference.

CaM activity was measured using CaM active enzyme continuous reaction spectrophotometry, which is based on activation of a CaM-dependent cyclic nucleotide phosphodiesterase44 (link). CaM activity assays were performed using a Plant CaM Active Enzyme Continuous Reaction Spectrophotometry Assay Kit (GENMED, USA). A 50 ml culture at OD₇₀₀ = 1 (about 1.3 × 10⁷ cells ml^–1) was harvested by centrifugation at 3,000 g for 3 min at room temperature, and the algal pellets were washed with 3 ml Reagent A in Kit, and centrifugation was repeated. The washed algal pellets were resuspended in pre-cooling 1 ml of Reagent B. The suspension was sonicated at 200 W for 30 min (3 s working time and 3 s interval in a cycle) in an ultrasonic cell disruptor at 4 °C. The suspension was boiled for 90 s and then cooled for 5 min in ice. Then supernatant was collected by centrifugation at 10,000 g for 5 min at 4 °C. The supernatants were used for CaM activity analysis directly according to the manufacturer’s instructions. Protein content was assayed using a BCA Protein Quantification Kit (TIANGEN, China). CaM activity (μg mg⁻¹ protein) was related to the amount of protein in the cell homogenate and expressed as milligrams of CaM activity per milligram of cell protein.

ATP synthetase activity assays were performed using an ATP Synthetase Assay Kit (GENMED, USA). A 100 ml culture at OD₇₀₀ = 1 (about 1.3 × 10⁷ cells ml^–1) was harvested by centrifugation at 3,000 g for 3 min at room temperature, and the algal pellets were washed with 1 ml Reagent A in Kit, and centrifugation was repeated. The washed algal pellets were resuspended in pre-cooling 4 ml of Reagent B. The suspension was sonicated at 200 W for 30 min (3 s working time and 3 s interval in a cycle) in an ultrasonic cell disruptor at 4 °C, and the supernatant was collected by centrifugation at 1,600 g for 10 min at 4 °C. Then supernatant was centrifuged at 10,000 g for 60 min at 4 °C to collect sediment. The collected sediment was dissolved into 200 μl Reagent B and used for the activity assays of the ATP synthetase in thylakoid membrane according to the manufacturer’s instructions. Protein content was assayed using a BCA Protein Quantification Kit (TIANGEN, China). ATP synthetase activity (U mg⁻¹ protein) in thylakoid membrane was related to the amount of protein in the chloroplast homogenate and defined as the amount of enzyme that caused per micromoles NADH oxidation per minute per milligram of protein at 37 °C and pH 8.0.

The total protein was extracted from 4 WT and 4 MT Chahua chickens’ ovarian tissue randomly selected using a rapid cell lysate kit (Solarbio, Beijing, China). Protein concentration was quantified using the BCA protein quantification kit (Tiangen, Beijing, China). The proteins were separated by SDS-PAGE gel electrophoresis under denaturing and non-reducing conditions and transferred to a polyvinylidene fluoride membrane (PVDF). After the addition of a 5% bovine serum albumin/TBST solution, the membrane was agitated for 1 h. Mouse monoclonal antibody anti-MMP-13 (Thermo, New York, NY, USA, 1:1000) was added, and the membrane was agitated for 30 min, followed by overnight incubation at 4 °C. The membrane was washed 3 times with TBST for 10 min each, followed by the addition of goat anti-mouse immunoglobulin G antibody (Thermo, New York, NY, USA, 1:1000) and incubation in a shaker for 1 h. The 3 TBST washes were performed again for 10 min each. The PVDF membrane was exposed to chemiluminescence to obtain the experimental results. The intensities of the blots were quantified using ImageJ software [19 (link)].

Mitochondria were isolated as previously described [29 (link)]. The minced blood-free atrial tissues were homogenized six times (0–4 °C) using a manual glass homogenizer. Subsequently, the homogenates were centrifuged at 1000 × g for 10 min to collect the supernatant, followed by centrifugation at 10,000 × g for 10 min. The main component of the sediment was mitochondrial precipitation, which was suspended in 0.5 mL of medium (HEPES 0.143 g, potassium chloride 1.928 g, EDTA Na2 0.037 g, KH2PO4 0.054 g, bovine serum albumin 0.2 g, distilled water 200 mL, pH 7.4). The mitochondrial isolation was best completed within 1 h after euthanasia. Mitochondrial protein content was determined using the bicinchoninic acid (BCA) protein quantification kit (TIANGEN, Beijing, China). Western blot analysis was implemented to assess the expression levels of mitochondrial biogenesis-, division-, and fusion-related proteins in the left atrial tissues.