ALP activity assay was performed according to the manufacturer’s instructions (Jiancheng, Nanjing, People’s Republic of China). Following a wash with PBS, the cells were lysed with 1% Triton X-100 (Beyotime, Shanghai, People’s Republic of China) on each well. The lysate was collected with the aid of a cell scraper and centrifuged at 12,000 rpm for 15 minutes at 4°C to remove cell debris. Total protein content in the lysate was measured using a bicinchoninic acid assay kit (Jiancheng). The absorbance of samples was recorded at a wavelength of 562 nm using a microplate reader. Cell lysate was prepared as mentioned earlier, which proceeded for 15 minutes at 37°C, and then a developer was added. The absorbance was measured at 520 nm with a microplate reader using p-nitrophenol phosphate as the standard. All results were normalized by the total intracellular protein content, and thus expressed as units/g protein. One unit of enzyme is defined as the amount of enzyme that converts 1 g of protein to 1 mg of p-nitrophenol phosphate in 15 minutes at 37°C.
Bicinchoninic acid assay kit
Manufactured by Nanjing Jiancheng
Sourced in China
The Bicinchoninic acid (BCA) assay kit is a colorimetric detection method for quantifying the total protein concentration in a sample. The kit utilizes the bicinchoninic acid compound, which reacts with proteins to produce a purple-colored complex that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Beyotime (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Ecl chemiluminescence detection kit, by Advansta (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail mixture, by Merck Group (1 mentions)
8 protocols using bicinchoninic acid assay kit
1
ALP Activity Assay Protocol
2
Cerebral Oxidative Stress Biomarkers
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The brain tissue were homogenized with 9 times the volume of PBS on ice and then centrifuged to prepare homogenates. The contents of malondialdehyde (MDA) and glutathione (GSH) as well as the activities of SOD and GSH-Px in the cerebral homogenates were measured following the respective manufacturer’s protocols (Nanjing Jiancheng Bio-Engineering Co., Ltd., Nanjing, China). Protein contents in the cerebral homogenates were determined using the bicinchoninic acid assay kit (Nanjing Jiancheng Bio-Engineering Co., Ltd., Nanjing, China).
3
Oxidative Stress Biomarkers in Liver
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Partial hepatic tissues were homogenized on ice. The liver homogenates were subjected to measure the level of MDA and GSH, the activities of SOD and GSH-Px according to their respective manufacturer’s instructions (Nanjing Jiancheng Bio-Engineering Co., Ltd., Nanjing, China). The protein contents in liver homogenates were measured using the bicinchoninic acid assay kit (Nanjing Jiancheng Bio-Engineering Co., Ltd., Nanjing, China).
4
Intestinal Mucosa Homogenization and Protein Analysis
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The small intestinal mucosa (jejunum and ileum) samples were homogenized with an ice-cold buffer containing 0.86% sodium chloride buffer (w/v 1:9). The supernatant was collected by centrifugation at 3,500 × g for 10 min at 4°C and stored for further analysis. The protein contents of the intestinal homogenates were determined by using a bicinchoninic acid assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
5
Protein Expression Analysis in Liver Tissue
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The liver tissues were homogenized, then centrifugated at 14,000 rpm for 20 min. The protein concentration in the supernatants was measured using the bicinchoninic acid assay kit (Nanjing Jiancheng Bio-Engineering Co., Ltd., Nanjing, China). The proteins were separated by 10% SDS-PAGE, then transferred to a nitrocellulose membrane by electroblotting. After blocking with TBS-T (20 mM Tris-HCl, 0.1% Tween 20, and 137 mM NaCl) containing 5% non-fat dry milk for 1 h at room temperature, the membrane was incubated with primary antibodies of cleaved caspase-3 (Cell Signaling Technology, Boston, MA, USA), bcl-2, and β-actin (Santa Cruz, Dallas, TX, USA) for 2 h, and then incubated with a secondary antibody horseradish peroxidase-conjugated anti-rabbit IgG/ anti-mouse IgG (Santa Cruz, Dallas, TX, USA) for 2 h at room temperature. Finally, the membrane was treated with the reagents in an electrogenerated chemiluminescence (ECL) chemiluminescence detection kit (Advansta, Menlo Park, CA, USA) then scanned. The relative amount of caspase-3 and bcl-2 was corrected with the amount of β-actin in the same sample.
6
Cerebral Homogenate Analysis Protocol
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The brain tissue were homogenized with 9 times the volume of PBS on ice and then centrifuged to prepare homogenates. The contents of malondialdehyde (MDA) and glutathione (GSH) as well as the activities of SOD and GSH-Px in the cerebral homogenates were measured following the respective manufacturer's protocols (Nanjing Jiancheng Bio-Engineering Co., Ltd., Nanjing, China). Protein contents in the cerebral homogenates were determined using the bicinchoninic acid assay kit (Nanjing Jiancheng Bio-Engineering Co., Ltd., Nanjing, China).
7
Cerebral Oxidative Stress Biomarkers
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The brain tissue homogenates were made by homogenizing each cerebral cortex tissue with 9 times volume of PBS on ice, then centrifuging. The contents of malondialdehyde (MDA) and glutathione (GSH) as well as the activities of SOD and GSH-Px in the cerebral homogenates were measured according to their respective manufacturer's instructions (Nanjing Jiancheng Bio-Engineering Co., Ltd., Nanjing, China). The protein contents in cerebral homogenates were detected using the bicinchoninic acid assay kit (Nanjing Jiancheng Bio-Engineering Co., Ltd., Nanjing, China).
8
Western Blot Analysis of TGF-β1 Signaling
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The tissues were lysed in a buffer containing 40 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM DTT, 1% Triton X-100, 2 mM Na 3 VO 4 , 10 mM NaF, and 10 mM sodium pyrophosphate supplemented with a protease inhibitor cocktail mixture (Sigma). Protein content was measured with a bicinchoninic acid assay kit (Jiancheng Institute of Biotechnology, Nanjing, China). Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis and proteins were transferred onto an Immobilon-P membrane. After a blocking procedure, the membrane was incubated overnight with anti-transforming growth factor (TGF)-β1, p-Smad2/3 or anti-GAPDH antibody at 4°C, followed by incubation with a secondary antibody coupled to horseradish peroxidase.
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