The total protein of each well was obtained by the radioimmunoprecipitation assay lysis buffer after 1, 6, and 24 h of cocultivation. The protein concentration was measured using bicinchoninic acid assays (Solarbio, China). Different samples were assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polyvinylidene difluoride (PVDF) membranes (Millipore Immobilon-P, Darmstadt, Germany). PVDF membranes were then blocked with PBS containing 5% nonfat dry milk and 0.05% Tween 20 for approximately 2 h.[12 (link)] The PVDF membranes were probed with primary antibodies (TLR2: rabbit-derived mouse antibody, SAB1300199, Sigma, USA; β-Actin: mouse-derived antibody, TA-09, Zsbio, China) both at a dilution of 1:1000 overnight in 4°C and then incubated with the corresponding secondary antibodies conjugated to horseradish peroxidase (TLR2: rabbit antibody IgG, zb-2301, Zsbio, China; β-Actin: mouse antibody IgG, A4416, Sigma, USA) at a dilution of 1:5000 and 1:1000, respectively, for 2 h at room temperature. Protein bands were exposed and then photographed, and the gray value was measured with Gel-Pro Analyzer Software (Media Cybernetics, Rockville, MD, USA). The TLR2 level was standardized by the gray value ratio of TLR2/β-Actin.
Bicinchoninic acid assay
Manufactured by Solarbio
Sourced in China
The bicinchoninic acid (BCA) assay is a colorimetric detection and quantification method used to measure the total protein concentration in a sample. It relies on the reduction of Cu2+ to Cu+ by proteins in an alkaline medium, followed by the chelation of the Cu+ ions with bicinchoninic acid, resulting in a purple-colored complex that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Solarbio (4 mentions)
Anti gapdh, by Proteintech (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
A4416, by Merck Group (1 mentions)
Zb 2301, by ZSGB-BIO (1 mentions)
Gel pro analyzer software, by Media Cybernetics (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Ta 09, by ZSGB-BIO (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay (ripa), by Solarbio (1 mentions)
X ray film, by Carestream (1 mentions)
Anti ctgf, by Proteintech (1 mentions)
Anti fibronectin, by ABclonal (1 mentions)
Anti collagen 1, by Proteintech (1 mentions)
Anti mmp 9, by Affinity Biosciences (1 mentions)
Anti timp 1, by Affinity Biosciences (1 mentions)
Anti collagen 3, by Proteintech (1 mentions)
Anti α sma, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Proteintech (1 mentions)
Amicon ultra 15, by Merck Group (1 mentions)
22 protocols using bicinchoninic acid assay
1
Measuring TLR2 Expression in Coculture
2
Protein Extraction and Western Blot Analysis
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After cell culture meets the requirements, total protein was extracted using radio immunoprecipitation assay (RIPA, Solarbio, China) lysis buffer. Then measured with bicinchoninic acid assays (Solarbio, China) with bovine serum albumin as a standard. Gel electrophoresis was then performed, 5% milk was incubated for 2 h and the primary antibody was incubated at 4°C overnight. The second antibody was incubated for 2 h at room temperature and then the membrane was imaged using enhanced chemiluminescence reagent (Thermo, China) and X-ray film (Carestream, China).
3
Extracellular Matrix Protein Analysis
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Uterine tissue and cultured hEnSCs were lysed using radioimmunoprecipitation (RIPA) buffer, and protein concentrations were determined using a bicinchoninic acid assay (Solarbio, China). Cell and tissue samples were electrophoresed on sodium dodecyl sulfate polyacrylamide gel followed by transfer to PVDF membranes. After blocking with 5% skim milk, the membranes were incubated with anti-Collagen I (1 : 2000; Proteintech, China), anti-Collagen III (1 : 1500; Proteintech, China), anti-CTGF (1 : 800; Proteintech, China), anti-Fibronectin (1 : 800; ABclonal, China), anti-α-SMA (1 : 1000; Abcam, UK), anti-MMP-9 (1 : 1000; Affinity, China), anti-TIMP-1 (1 : 1000; Affinity, China), or anti-GAPDH (1 : 20000; Proteintech, China) polyclonal antibodies at 4°C overnight. On the following day, membranes were washed with TBS and Tween 20 (TBST) and then immunoblotted with HRP-conjugated secondary antibodies (Proteintech, China) for 1 h at room temperature. Expressions of each protein were determined using the enhanced chemiluminescence reagent (ECL) kit (Sparkjade Science Co., Ltd., China), and band densities were measured with using ImageJ software.
4
Extracellular Vesicle Isolation via Anion Exchange
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Extracellular vesicles were isolated using anion exchange chromatography, as reported [ 12 ]. Briefly, extracellular vesicles were first adsorbed into an anion exchange resin (Q Sepharose fast flow, GE Health Care Life Science) that was packed in an Econo-Pac column (Bio-Rad Laboratories). Extracellular vesicles were eluted with a 500 mM NaCl solution in phosphate-buffered saline (pH 7.4). The eluted extracellular vesicle solution was further desalted and concentrated via ultrafiltration (Amicon Ultra-15 with pore size of 100 kDa; Merck). The concentration of extracellular vesicles was determined by bicinchoninic acid assay (Solarbio). The purified extracellular vesicles were stored at -80°C.
5
Western Blot Assay for CEP55 Quantification
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For western blot assays, total protein was extracted from cells after the indicated treatment. Cells were washed for three times in cold phosphate-buffered solution (PBS) and lysed in lysis buffer (Solarbio, China) containing 1% PhosSTOP phosphatase inhibitor (Roche, Switzerland). Bicinchoninic acid assay (Solarbio, China) was applied to measure protein concentration. Primary antibodies against CEP55 were purchased from Abcam. βactin was applied as the loading control. Primary antibody against β-actin was obtained from Zhongshan Goldenbridge Biotechnology Company. Horseradish peroxidase-conjugated goat-anti-mouse and goat-anti-rabbit antibodies (Zhongshan Goldenbridge Biotechnology Company, SPN-9002 and SPN-9001, respectively) were involved as secondary antibodies.
6
Isolation and Characterization of ADMSCs-Derived Extracellular Vesicles
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EVs were isolated from supernatants of ADMSCs (5 × 106) by ultracentrifugation. A concentrated solution was enriched in supernatants via sucrose density gradient centrifugation, as previously described (Jiang et al., 2018 (link); Kholia et al., 2018 (link)). The prepared sucrose solution and concentrated solution were successively added to an ultracentrifugation tube at a ratio of 1:8, and centrifuged at 110,000 × g at 4°C for 70 min to collect the heavy water layer. An appropriate amount of phosphate‐buffered saline (PBS) was added to resuspend the precipitate, which was centrifuged at 110,000 × g at 4°C for another 70 min. Collected precipitates were resuspended in 50–100 μL PBS.
EVs were characterised by expression of typical marker proteins and protein concentrations were determined by bicinchoninic acid assay (Beijing Solarbio Science & Technology Co., Ltd). Morphological structures were observed under transmission electron microscopy (TEM), and particle size distribution was analysed via Nanoparticle Tracking Analysis (NTA). Surface markers including exosomal markers (CD63, CD9, Tsg101) were analysed by western blotting.
EVs were characterised by expression of typical marker proteins and protein concentrations were determined by bicinchoninic acid assay (Beijing Solarbio Science & Technology Co., Ltd). Morphological structures were observed under transmission electron microscopy (TEM), and particle size distribution was analysed via Nanoparticle Tracking Analysis (NTA). Surface markers including exosomal markers (CD63, CD9, Tsg101) were analysed by western blotting.
7
Western Blot Analysis of Claudin-3
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Tissue was first lysed using RIPA buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.). The protein concentration of the mucosal samples was detected by bicinchoninic acid assay (Beijing Solarbio Science & Technology Co., Ltd.). An equal amount of protein (40 µg) of each sample was loaded onto 12% polyacrylamide gels for electrophoresis and then transferred to a PVDF membrane. After blocking at 25˚C with 5% fat-free milk for 1-2 h, the membranes were incubated with anti-claudin-3 antibody (cat. no. ab15102; 1:200; Abcam) and β-actin (1:5,000; cat. no. 66009-1-lg; ProteinTech Group, Inc.) at 4˚C overnight. After incubation at 25˚C with horseradish peroxidase-conjugated secondary antibody (cat. no. SA00001-2, dilution 1:2,000; ProteinTech Group, Inc.) for 2 h, the membranes were visualized by Mini Chemi 610 electrochemiluminescence (Beijing Sage Creation Science Co., Ltd.) and the gray value was analyzed using Lane ID software 5.0 (Beijing Sage Creation Science Co, Ltd.).
8
Isolation and Characterization of hAFSC-Derived Exosomes
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The hAFSCs were grown to 80% confluency in DMEM containing 10% FBS, then replaced with serum-free medium (Biological Industries, Israel) and cultured for 48 h. The culture suspension was collected and centrifuged as follows: 2,000g for 10 min (removing dead cells) → 10,000g for 30 min (removing debris) → 100,000g for 70 min (collecting exosomes). The exosomes were then washed three times with PBS. The protein concentration of hAFSC-exo was measured using the bicinchoninic acid assay (Solarbio, China). The hAFSC-exo product was identified using transmission electron microscopy, NanoSight NS300 (Malvern Instruments, United Kingdom), and exosomal markers CD9, CD63, and TSG101 (Beyotime, China) using western blot analysis. The exosomes were stored at −80°C for subsequent studies.
9
Proteomic Analysis of Mouse Myocardium
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As described previously, the mice hearts were harvested and quickly washed with PBS to remove the blood, and protein samples from the myocardium and cells were extracted. The heart tissue was cut into 2 mm thickness and then the protein was extracted from the left ventricular samples using a RIPA system. Then protein concentration was determined by bicinchoninic acid assay (Solarbio co, LTD, Shanghai, China). Extracted protein was separated by 8–12% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Millipore, MA, USA). After being blocked and dissolved, the membranes were incubated with the primary antibodies against LC3II, P62, mTOR, p-mTOR, BAX, BCL-2, p-Akt, Akt, and GAPDH overnight at 4 °C. The protein bands were detected by a chemiluminescent system, and the bands were scanned and quantified by densitometric analysis using Image Lab 3.0.
10
Protein Extraction from Ovarian Cancer Tissue
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Total protein was extracted from tumor tissue using radio-immunoprecipitation assay (RIPA) buffer (Solarbio Biotech Corp., Beijing, China) following the manufacturer instructions. Briefly, ovarian cancer tissues were lysed for use in antibody array analysis in RIPA buffer containing 1% phenylmethylsulfonyl fluoride (Solarbio Biotech Corp.), and homogenized using a microtissue grinder (Kimble Chase, Thailand). The supernatants were collected after centrifugation at 14,000 g and 4°C for 15 min. The protein concentration was determined using a bicinchoninic acid assay (Solarbio Biotech Corp.).
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