The intracellular GSH content was determined by using a microplate-reader method with a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). LTEP-a-2 cells were inoculated into 6-well plates at 106cells/well and then exposed to different concentrations of ZnO NPs (0, control; 0.01, 0.05, and 0.25 μg/mL) for 4 h. Cells were then harvested and washed with PBS. The content of GSH was assayed by measuring the absorbance of cell extract at 412 nm using a microplate reader, calculated according to a standard curve, and normalized by the protein concentration detected using the Bradford method (Sangon, Shanghai, China) [22 (link)].
Bradford method
Manufactured by Sangon
Sourced in China
The Bradford method is a spectrophotometric analytical procedure used for the quantitative determination of total protein concentration in a sample. It is a widely used technique in biochemistry and molecular biology laboratories.
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Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Ecl kit, by Merck Group (3 mentions)
Image lab, by Bio-Rad (2 mentions)
Ecl reagent, by Ipsen (1 mentions)
Ripa cell lysis buffer, by Beyotime (1 mentions)
Lc3 1 2, by Cell Signaling Technology (1 mentions)
Gapdh, by Abcam (1 mentions)
α sma, by Santa Cruz Biotechnology (1 mentions)
Ecl chemiluminescent kit, by Merck Group (1 mentions)
Proliferating cell nuclear antigen (pcna), by Abcam (1 mentions)
Smad4, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Mouse anti collagen 1 antibody, by Abcam (1 mentions)
Ni nta beads, by Smart-Lifesciences (1 mentions)
Chemiluminescence kit, by Merck Group (1 mentions)
11 protocols using bradford method
1
Measurement of Intracellular GSH Content
2
Protein Expression Analysis in LX-2 Cells
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Total proteins were extracted from LX‐2 cells using standard methods. Protein concentration was quantified by Bradford method (Sangon, Shanghai, China). Protein samples were separated by SDS‐PAGE (8%‐12%), transferred onto PVDF membranes (Merck, Darmstadt, Germany) and blocked with 5% nonfat dry milk. Membranes were incubated with specific primary antibodies at 4°C overnight and then incubated with an appropriate second antibody at room temperature. A chemiluminescence (ECL) kit (Merck, Darmstadt, Germany) was used to detect target proteins. Protein bands were normalized to GAPDH, and protein expression was quantified by Image Lab of Bio‐Rad (Berkeley, California, USA).
3
Quantification of Autophagy Markers in HCAEC
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After co-culturing for 48 h, the cells were washed three times with cold PBS. To extract proteins, HCAECs were harvested and lysed in RIPA cell lysis buffer (Beyotime, Haimen, China) pre-chilled at 4 °C. Bradford method (Sangon Biotech, Shanghai, China) was employed to measure the concentration of total protein in the lysates. Approximately 20 µg lysate protein was loaded into each well in 12.5% sodium dodecyl sulfate-poly acrylamide gels, and then separated by electrophoresis at 100 V. Next, the proteins were transferred onto polyvinylidene fluoride membranes (PVDF, 0.22 µm; Bio-Rad, CA, USA). After blocking with 5% nonfat milk solution on a shaker at room temperature for 2 h, the membranes were incubated with a primary antibody against light chain 3 II/I (LC3 II/I) (1:1,000; Cat: 4108; Cell Signaling Technology (CST), Danvers, MA, USA) at 4 °C with gentle shaking for 14–16 h. After washing with PBS, the membranes were incubated with a secondary antibody (1:5,000; Cat: 7074; CST, USA) at room temperature for 1 h. Protein bands were detected using an electrochemiluminescence (ECL) reagent (EpiZyme, Shanghai, China) for development, and then visualized on an Image Quant LAS 4000 imaging system. The blots were also probed against GAPDH (1:5,000; Cat: ab9485; Abcam, Cambridge, MA, USA), which was used as an internal loading control.
4
Protein Expression Profiling in LX-2 Cells
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The total proteins were extracted from LX-2 cells and protein concentration was quantified by Bradford method (Sangon, China). Protein samples were separated by SDS-PAGE (8–12%), transferred onto PVDF membranes (Merck, Germany), and blocked with 5% nonfat dry milk. Membranes were incubated with primary antibodies against P-Rb, P-ERK, and Caspase 3 (antibody dilution is 1:2000, Cell Signaling Technology, USA). P27, SKP2, Rb and α-SMA were obtained from Santa Cruz Biotechnology (1:200 dilution, USA). And then incubated with an appropriate second antibody which is a peroxidase-labeled anti-rabbit (1:5000 dilution, Abcam, USA) or anti-mouse (1:10000 dilution, Abcam, USA) immunoglobulin at room temperature. The target proteins were detected using a Schemiluminescence (ECL) kit (Merck, Germany). Bands were normalized with GAPDH and protein expressions were quantified by Image J.
5
Protein Expression Profiling in LX-2 Cells
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Proteins from LX-2 were extracted using RIPA lysis buffer (Beyotime, China) and quantified by the Bradford method (Sangon, China). Then the proteins were separated on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 10% nonfat dry milk and then probed with primary antibodies against α-smooth muscle actin (α-SMA, Santa Cruz Biotechnology, USA), Smad4 (Santa Cruz Biotechnology, USA), PCNA (Abcam, USA) and glyceraldehyde phosphate dehydrogenase (GAPDH, Goodhere, China) at 4°C overnight. The membranes were then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Then the membranes were visualized with ECL-chemiluminescent kit (Merck, Germany).
6
Quantification of Protein Expression
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Total proteins were extracted from LX-2 cells using standard methods. Protein concentration was quantified by Bradford method (Sangon, Shanghai, China). Protein samples were separated by SDS-PAGE (8–12%), transferred onto Shanghai, PVDF membranes (Merck, Darmstadt, Germany) and blocked with 5% non-fat dry milk. Membranes were incubated with specific primary antibody at 4 °C overnight and then incubated with an appropriate second antibody at room temperature. A chemiluminescence (ECL) kit (Merck, Darmstadt, Germany) was used to detect target proteins. Protein bands were normalized to GAPDH and protein expression was quantified by Image J (National Institutes of Health, Bethesda, MD, USA).
7
Quantification of Collagen I and TGFβR I
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Total protein was extracted from LX-2 cells on ice using standard methods. Protein was quantified by Bradford method (Sangon, China). Each protein sample was separated by 10% SDS-PAGE, transferred onto a PVDF membrane (Merck, Germany) and blocked with 5% non-fat milk at room temperature for 2 hours. The membrane was then incubated with mouse anti-collagen I antibody (Abcam, USA) or rabbit anti-TGFβR I antibody (Santa Cruz, USA) overnight at 4°C, then with secondary antibodies (Santa Cruz, USA) for 1h at room temperature. Finally, protein bands were detected by a chemiluminescence (ECL) kit (Merck, Germany) and quantified using Image Lab (Bio-Rad, USA). For the above experiments, GAPDH was used as an internal control.
8
Characterization of Xylanase Enzymes from BL21 Strains
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Two BL21 strains with xylanase genes of PW-xyl9 or PW-xyl37 were selected for further characterization. The corresponding strains were incubated overnight in a shaker at 37 ℃ and 200 rpm; 4 mL of the overnight culture was then inoculated to 200 mL fresh LB medium in a 1 L shake flask for further culturing. Once the OD600 value of the two strains had reached 0.8, 200 mM IPTG was added, and the culture was cultivated at 20 ℃ for an additional 16 h. Cells were harvested and washed 3 × with PBS buffer, and were suspended in 20 mL PBS buffer with 1 mM phenylmethylsulfonyl fluoride (PMSF) which was protease inhibitor. The cells were disrupted by sonication for a total length of 15 min (Xiaomei, Kunshan, China), with setting parameters of 150 W, 3 s on, and 5 s off. The cell suspensions were centrifuged at 12,000 rpm at 4 ℃ for 20 min, and the supernatant was collected as crude enzyme. Magnetic beads (PuriMag, Xiamen, China) were used for protein purification, and the purified proteins obtained were concentrated in 10 kDa ultrafiltration tubes (Merck, Millipore, USA). The purity of proteins was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and their concentrations were measured with the Bradford method (Sangon Biotech, Shanghai, China).
9
Enzymatic Activity and Protein Quantification
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The filter paper hydrolyzing activity (FPase) and endo-beta-1,4-glucanase (CMCase) activity in culture supernatants were measured according to the International Union of Pure and Applied Chemistry (IUPAC) standard [27 (link)]. The extracellular protein concentration was determined by the Bradford method (Sangon Biotech Co., Ltd., Shanghai, China).
10
Purification of PW-pGH28-3 Protein from BL21 (DE3)
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The BL21 (DE3) strain harboring PW-pGH28-3 was incubated overnight in a shaker at 37 °C and 200 rpm. Then, 4 mL of the overnight culture was inoculated to 200 mL fresh LB medium (50 µg/mL kanamycin) in 1 L shake flask. When the OD600 reached 0.6–0.8, IPTG was added to reach 100 µM, and the cells were cultivated at 25 °C for another 6 h. Cells were harvested and washed three times with PBS, then suspended in 20 mL PBS with 1 mM phenylmethylsulfonyl fluoride (PMSF, protease inhibitor). The cells were disrupted by sonication. The cell lysates were centrifuged at 12,000 rpm at 4 °C for 20 min, and the supernatant was collected as crude enzyme solution. Ni NTA beads (Smart-Lifesciences, Jiangsu, China) were used for protein purification, and the purified PW-pGH28-3 protein was concentrated in 10 kDa ultrafiltration tubes (Merck Millipore, Darmstadt, Germany). The purity of PW-pGH28-3 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the concentration was measured with the Bradford method (Sangon Biotech, Shanghai, China).
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