Total proteins of the isolated exosomes and cells were extracted using a protein extraction kit, according to the manufacturer's protocols. Briefly, the samples were washed with phosphate-buffered saline and lysed in RIPA lysis buffer containing phenylmethylsulfonyl fluoride. The protein concentration was measured using a Bicinchoninic Acid protein assay kit (Sangon Biotech Co., Ltd., Shanghai, China), and 2 mg/ml bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) was used as the standard. Subsequently, 50 µg total protein was loaded in each lane. Proteins were separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The membranes were then blocked with 5% non-fat milk at room temperature for 1 h, and then incubated at 4°C overnight with a primary antibody. After washing, the blots were incubated with Alexa Fluor-conjugated goat-anti-rabbit antibody (1:10,000; cat. no. G-21234; Fermentas; Thermo Fisher Scientific, Inc.) at 37°C for 1 h. Proteins were visualized using an enhanced chemiluminescence kit, and images were analyzed using a Bio-Rad gel imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Bicinchoninic acid protein assay kit
Manufactured by Sangon
Sourced in China
The Bicinchoninic acid (BCA) protein assay kit is a colorimetric method used for the quantitative determination of total protein concentration. It relies on the reduction of Cu2+ to Cu+ by protein in an alkaline medium, with the chelation of the Cu+ ions by bicinchoninic acid to produce a purple-colored complex that exhibits a strong linear absorbance at 562 nm.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Protein extraction kit, by Sangon (2 mentions)
Protease inhibitor, by Thermo Fisher Scientific (2 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Enhanced chemiluminescence kit, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Anti muc1 antibody, by Abcam (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Image lab 3, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay (ripa), by Sangon (1 mentions)
Anti tgf β, by Cell Signaling Technology (1 mentions)
Anti p smad3, by Cell Signaling Technology (1 mentions)
Chemiluminescence kit, by Sangon (1 mentions)
Ripa buffer high, by Solarbio (1 mentions)
Goat anti mouse igg h l, by Proteintech (1 mentions)
Phenylmethylsulfonyl fluoride, by Solarbio (1 mentions)
19 protocols using bicinchoninic acid protein assay kit
1
Exosome Protein Extraction and Analysis
2
Quantitative Protein Analysis via SDS-PAGE
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The supernatant after cell lysis was collected with radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China). The concentration of proteins was measured by the bicinchoninic acid Protein Assay Kit (Sangon Biotech), and the same amount of protein was denatured with 5 × sodium dodecyl sulfate (SDS; Sangon Biotech) loading buffer in boiling water at 100°C for 5 min. To separate the proteins, SDS-polyacrylamide gel electrophoresis was used; then the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, and the PVDF membrane was incubated with 5% non-fat milk at room temperature for 1 h. Next, the anti-MUC1 antibody (1:1000; Abcam) and GAPDH (1:1000; Abcam) were added and incubated at 4°C overnight. Then the membrane was washed with Tris-buffered saline with Tween-20 three times; subsequently, the secondary antibody (HRP-labeled) was added and incubated at 20°C for 1 h. Finally, the protein was tested using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA).
3
Western Blot Analysis of TGF-β Signaling
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All transfected cells were seeded into 6-well tissue culture plates (1–2×106) at 37°C and lysed using 200 µl protein (RIPA; Sangon Biotech Co., Ltd., Shanghai, China). The protein concentration was determined using a Bicinchoninic Acid Protein Assay kit (Sangon Biotech Co., Ltd.). The proteins were separated using 12% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc.). Following blocking of the membranes using 5% nonfat milk in TBST, the membranes were incubated with anti-p-SMAD3 (#9520; dilution 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-TGF-β (#3709; dilution, 1:2,000; Cell Signaling Technology Inc.) and β-actin (#4970; dilution 1:2,000; Cell Signaling Technology, Inc.) antibodies at 4°C overnight. Following washing with TBST, the membranes were incubated with a goat anti-rabbit secondary antibody (#A16110; 1:5,000; Pierce; Thermo Fisher Scientific, Inc.) at 37°C for 1 h, and the blots were detected using an enhanced chemiluminescent reagent (#G-21234; Pierce; Thermo Fisher Scientific, Inc.) and quantified using Image Lab 3.0 (Bio-Rad Laboratories, Inc.).
4
Western Blot Analysis of Protein Expression
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Washing of the treated cells was carried out in phosphate buffered saline (PBS) and lysed in RIPA buffer (high) (Solarbio, Beijing, China), which contained 1 mM phenylmethylsulfonyl fluoride, Solarbio. Calculation regarding protein concentration was done utilizing the bicinchoninic acid protein assay kit (Sangon Biotech). Equivalent amounts of whole protein extract (30 µg) were electrophoresed on SDS‐polyacrylamide gels and then transferred to polyvinylidene difluoride membranes (pore size: 0.22 µm) using the Yeasen Blot‐transfer unit. After blocking with 5% milk, incubated the membranes overnight with specific primary antibodies against Survivin (Mouse/IgG1; 1:3,000; Proteintech), glyceraldehyde‐3‐phosphate dehydrogenase (Mouse/IgG2b; 1:50,000; Proteintech), His (Mouse/IgG1; 1:10,000; Proteintech), Flag (Rabbit/IgG; 1:10,000; Bioworld), HA (Human influenza hemagglutinin) (Rabbit/IgG; 1:3,000; Bioworld), EGFP (Mouse/IgG1; 1:2000; Leading Biology) at 4°C. The membranes were later incubated with horseradish peroxidase‐conjugated secondary antibody (Goat Anti‐Rabbit IgG (H+L) or Goat Anti‐Mouse IgG (H+L); 1:6000; Proteintech) for 2 h on a horizontal oscillator at room temperature. Products were generated on film by the chemiluminescence kit purchased from Sangon Biotech and quantitated by ImageJ software.32 , 33 , 39
5
Western Blot Analysis of MMP1 Protein
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Cultured cells were washed 3 times with PBS and lysed in phenylmethanesulfonyl fluoride (Sangon Biotech Co., Ltd., Shanghai, China) on ice for 20 min. The lysates were centrifuged at 15,000 × g at 4°C for 10 min and the supernatants were collected. The protein concentration was measured using a Bicinchoninic Acid protein assay kit (Sangon Biotech Co., Ltd.), with 2 mg/ml BSA (Gibco; Thermo Fisher Scientific, Inc.) as the standard. Subsequently, 50 µg proteins were separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Merck KGaA). The membranes were blocked in 5% nonfat dried milk at room temperature for 1 h, and then incubated at 4°C with primary antibodies (anti-MMP1 antibody; 1:10,000; cat. no. sc-58377; Santa Cruz Biotechnology, Inc., Dallas, TX, USA and anti-β-actin antibody, 1:10,000; cat. no. 115035003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) overnight. Following washing with Tris buffered saline with Tween-20 (1:1,000) 4 times, the blots were incubated with horseradish peroxidase-conjugated goat-anti-rabbit antibody (1:5,000; cat. no. 111035047; Jackson ImmunoResearch Laboratories, Inc.) at 37°C for 1 h, and then developed with enhanced chemiluminescence detection reagents (Merck KGaA). Visualization was performed with a gel imaging analysis system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
6
Western Blot Analysis of Protein Expression
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Cell lysates were prepared in 50 μl of radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) containing 1 mmol phenylmethanesulfonyl fluoride (PMSF) (Beyotime, China). Protein concentrations were quantified using a bicinchoninic acid protein assay kit (Sangon Biotech, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 10% (0.1 g/ml)) was used to separate proteins, which were then electrotransferred onto polyvinylidene difluoride membranes (Millipore, USA). Tris-buffered saline containing 0.05% Tween 20 (TBST) and 5% nonfat milk was used to block the blots before they were probed with primary antibodies, mouse monoclonal anti-N IgG and rabbit monoclonal anti-NF-κB IgG (CST, USA) at 1:500 (v/v) dilution and rabbit monoclonal anti-β-actin IgG (Hua’an, China) at 1:2000 (v/v) dilution. Blots were subsequently washed with TBST and incubated for 1 h with secondary antibodies, goat anti-mouse horseradish peroxidase (HRP)-labeled antibody, and goat anti-rabbit HRP-labeled antibody (Sangon Biotech, China). West Pico chemiluminescent substrate (Thermo, USA) was used to visualize the Western blotting results. The Gel 3100 chemiluminescent imaging system (Sagecreation, China) was used to capture images.
7
Purification and Characterization of PuM90-RBD
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Due to the difficulty of expressing and purifying intact PuM90, the CDSs of the PuM90 RNA binding domain (PuM90-RBD) and the PuM90 RNA binding domain with three amino acid residues mutated in all eight repeats (PuM90-RBDAAA) were separately inserted into the pGEX-4T-2 vector (containing the glutathione S-transferase [GST] tag; GE Healthcare Life Science) for in vitro assays. The plasmids (GST empty vector, GST-PuM90-RBD, and GST-PuM90-RBDAAA) were transformed into E. coli strain BL21 (DE3). A 500-mL culture of E. coli BL21(DE3) cells was grown at 37°C to an optical density at 600 nm of 0.5, after which gene expression was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (Sigma) for 4 h at 28°C. After lysing of cells, the recombinant proteins were purified on glutathione Sepharose beads (Sangon Biotech, Shanghai, China) and eluted with 5 mM glutathione dissolved in Tris-buffered saline. The concentration of purified proteins was determined using a bicinchoninic acid protein assay kit (Sangon Biotech). Protein purity was assessed via SDS-PAGE.
8
Whole Cell Protein Extraction and Western Blot Analysis
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Western blot analysis was performed on whole cell extracts obtained by the direct dissolution of cells using a whole cell protein extract reagent (M-PER; Pierce Biotechnology, Inc., Rockford, IL, USA) according to the manufacturer’s instructions. Protein concentrations were then determined using a bicinchoninic acid protein assay kit and bovine serum albumin [both Sangon Biotech (Shanghai) Co,. Ltd., Shanghai, China] was used as a control. Next, the proteins (40 μg/lane) were separated on 12% SDS-PAGE gels (Beyotime) and transferred onto polyvinylidene difluoride membranes (Bio-Rad). The membranes were then blocked with 5% non-fat milk in phospshate-buffered saline with Tween [PBST; 0.2% Tween-20 in PBS (pH 7.6); Beyotime] and incubated with the primary antibodies (1:1,000) for 18–24 h at 4°C. The membranes were subsequently incubated with the secondary antibodies conjugated to HRP (1:5,000) for 1 h at 37°C. Finally, the protein bands were visualized using an enhanced chemiluminescence western blot detection kit (Pierce Biotechnology, Inc.).
9
Protein Quantification and Western Blot Analysis
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The total concentrations of proteins in small intestinal tissues were determined using the bicinchoninic acid protein assay kit (Sangon Biotech Co., Shanghai, China). The protein concentration of each sample was 4 μg/μL. The total protein (50 μg) was electrophoresed on 8 or 10% sodium dodecyl sulfate polyacrylamide gel. The separated proteins were transferred to a PVDF membrane (Millipore Co., Billerica, MA, USA). Block the membrane for non-specific binding proteins in 5% skimmed milk at room temperature 20–24°C for 1 h, and then incubated overnight at 4°C with Anti-Lysozyme antibody (1:1,000; Abcam, Cambridge, MA, UK), Anti-occludin (1:1,000; Abcam, Cambridge, MA, UK), Anti-claudin-1(1:1,000; Abcam, Cambridge, MA, UK), Anti-secretory phospholipase A2 antibody rabbit (1:1,000; Abcam, Cambridge, MA, UK), and Anti-GAPDH antibody (1:10,000; Santa Cruz Biotechnology, MA, USA). Subsequently, the membrane was washed three or five times in Tris-buffered saline containing Tween-20 and incubated with appropriate species-specific secondary antibodies at 20–24°C for 1 h at room temperature. Use chemiluminescence detection reagents and expose them to Kodak XAR film. Using the GAPDH bands as an internal control, all band densities were analyzed with ImageJ software.
10
Quantifying TGF-β1 in Lung Tissue
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The protein in the right lungs was extracted using a Protein Extraction kit (Shanghai Sangon Biotechnology, Shanghai, China). Protein concentrations were determined using a Bicinchoninic Acid Protein Assay kit (Shanghai Sangon Biotechnology). Concentrations of transforming growth factor (TGF)-β1 in lung homogenates were quantified with a Mouse TGF-β1 protein extraction and enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer's instructions. Optical density values were detected using a Microplate Reader (iMark; Bio-Rad, Hercules, CA, USA) at 450 nm.
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