VSMCs were treated with 50 µM DHA and transfected with the microRNA-155 mimics and inhibitors before harvesting for protein isolation. Proteins were isolated in the protein extraction buffer (Beyotime Institute of Biotechnology). The protein concentration was measured by BCA method. Protein samples (20 µg/lane) were separated by SDS-PAGE on 10% gels and transferred to a nitrocellulose membrane. The membranes were incubated with 5% fat-free milk in TBS with Tween® 20 for 1 h at room temperature and with primary antibodies overnight at 4°C. The PVDF membranes were washed with TBST buffer and incubated with peroxidase-conjugated specific secondary antibodies from Abcam [Ki67 (1:5,000; cat. no. ab15580), Socs1 (1:4,000; cat. no. ab62584) and actin (1:3,000; cat. no. ab6276)] with shaking for 1 h. The enhanced chemiluminescence solution (Sigma-Aldrich; Merck KGaA) was the prepared in a dark room, and the exposure time of the film was determined according to the chemiluminescence intensity. Densitometric analysis was performed using Image Lab software (version 3.0; Bio-Rad Laboratories, Inc.).
Enhanced chemiluminescence solution
Manufactured by Merck Group
Sourced in United States, Germany
Enhanced chemiluminescence solution is a laboratory reagent used to detect and quantify proteins in Western blot analysis. The solution contains chemicals that produce a luminescent signal when they interact with the target protein, allowing for visualization and measurement of protein levels.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (15 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Ab8226, by Abcam (4 mentions)
B27 supplement, by Thermo Fisher Scientific (4 mentions)
Celltiter 96 aqueous one solution cell proliferation assay kit, by Promega (4 mentions)
Neurobasal media, by Thermo Fisher Scientific (4 mentions)
Ab6721, by Abcam (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Cadmium chloride, by Merck Group (3 mentions)
Akt inhibitor x, by Santa Cruz Biotechnology (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Poly d lysine 4 6 diamidino 2 phenylindole dapi, by Merck Group (3 mentions)
Image lab software, by Bio-Rad (2 mentions)
Ab181602, by Abcam (2 mentions)
Bradford protein assay, by Bio-Rad (2 mentions)
Ab8227, by Abcam (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
2 7 dichlorofluorescein, by Merck Group (2 mentions)
57 protocols using enhanced chemiluminescence solution
1
MicroRNA-155 Regulation in VSMCs
2
Immunoprecipitation and Western Blot Analysis
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For immunoprecipitation, CD4+ T cells were resuspended in 2 mL of ice-cold lysis buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton, 2 mM Na3VO4, 5 mM NaF, 1 mM EDTA, and protease inhibitor cocktail tablet [Roche]) and incubated for 20 min on ice. Lysates were cleared by centrifugation at 18,000 × g for 15 min at 4°C, and the dynein intermediate chain (DIC) was subjected to immunoprecipitation from cleared lysates for 2 hr at 4°C with 15 μL of protein G-Sepharose resin coated with 12 μg of polyclonal rabbit anti-DIC antibodies. The resin was washed three times and incubated for 10 min at 95°C with Laemmli buffer. For p53 analysis, CD4+ T cells were stimulated with 10 μg/mL of anti-CD3 and 2 μg/mL of anti-CD28 antibodies for 24 and 48 hr and were suspended in ice-cold lysis buffer after each time point. Proteins were resolved by SDS-PAGE and transferred to PVDF membranes according to standard protocols. Membranes were blocked with 5% milk in Tris-buffered saline containing Tween at 0.05% for 1 hr at room temperature before being incubated with primary antibodies at 4°C overnight. After washing, membranes were incubated with secondary antibodies for 1 hr at room temperature. Subsequently, membranes were incubated with enhanced chemiluminescence solution (Sigma) for 5 min in the dark, and luminescence was captured with a Bio-Rad XRS+ imager.
3
Antioxidant Screening of Biomolecules
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λ-DNA was purchased from Bangalore Genei, India. Agarose, H2O2, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), dimethyl sulfoxide (DMSO) were purchased from Sisco Research Laboratories. FeSO4·7H2O, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, ascorbic acid, Tris base, ethidium bromide (EtBr), ethylenediaminetetraacetic acid (EDTA), Enhanced Chemiluminescence solution (ECL), vitexin, isovitexin and quercetin were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). Polyvinylidene fluoride membrane (PVDF) was purchased from pall corporation, (New York, USA). BCA protein assay kit was purchased from Thermo Fisher Scientific (Massachusetts, USA). All other chemicals used were of analytical grade.
4
Western Blot Analysis of Epigenetic Regulators
5
Western Blot Analysis of SPHK1 Protein
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To detect protein expression, TPC-1 cells that had been transfected for 24 h were washed in PBS and lysed with radioimmunoprecipitation assay buffer (50 mM TrisHCl pH 7.4; 150 mM NaCl; 2 mM EDTA; 1% NP-40; 0.1% SDS). The protein concentrations were determined using a bicinchoninic acid protein quantitation assay kit (Beyotime Institute of Biotechnology). Protein (20 µg) were separated by 10% SDS-PAGE and transferred onto a PVDF nylon membrane (Merck KGaA) at 4°C. Membranes were blocked for 30 min in 5% non-fat powdered milk at room temperature. After washing with TBS/Tween-20 (TBST) three times for 5 min each, the membranes were incubated with the anti-SPHK1 primary antibody (1:100; cat. no. ab71700; Abcam) for 8 h at 4°C. The membranes were then washed three times with TBST for 10 min each to remove free primary antibody. Mouse anti-GAPDH antibody (1:1,000 dilution; cat. no. ab8245; Abcam) was used as the loading control. After further washing, the membranes were incubated in a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:1,000; cat. no. sc2004; Santa Cruz Biotechnology, Inc.) solution for 1 h at room temperature. All blots were visualized using an enhanced chemiluminescence solution (Merck KGaA) and analyzed using Quantity One Protein Analysis software version 4.6.7 (Bio-Rad Laboratories, Inc.).
6
Western Blot Analysis of Protein Expression
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Cultured cells were collected by trypsinization, and then lysed with enhanced radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor (Boster, Wuhan, China), and then the protein concentration was determined by bicinchoninic acid (BCA) protein quantitative kit (Boster). The protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane and blocked with 5% bovine serum albumin at room temperature. Then diluted primary rabbit antibodies (SIRT1, ab220807; KLF4, ab106629; MMP2, ab97779; β-actin, ab8227; 1: 500; Abcam, Cambridge, MA, USA) were added into the membrane, followed by incubation at 4°C overnight. Next, horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (ab205719; 1: 2000; Abcam) was used to incubate the membrane for 1 hour at room temperature. The enhanced chemiluminescence solution (EMD Millipore, Bedford, MA, USA) was used to incubate the membrane at room temperature for 1 minute, after which it was then sealed with the plastic wrap, followed by X-ray film exposure for 5-10 minutes, development and fixation. Image J software was used to quantify the gray intensity of each band in Western blot analysis image, and β-actin was used as internal reference.
7
Western Blot Analysis of Apoptosis Markers
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The MSCs were seeded into a 60 mm petri dish, at a density of 10×105 cells/dish. The total protein was extracted using radioimmunoprecipitation buffer (EMD Millipore), supplemented with PMSF. The cells were sonicated briefly and centrifuged at 10,000 x g at 4°C. The protein concentration was measured using the BCA Protein Assay kit, according to the manufacturer's instructions. Equal samples of protein (20 µg) were separated by 12% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated with blocking solution (Beyotime Institute of Biotechnology, Jiangsu, China) at room temperature for 2 h, and then incubated with the following primary antibodies: Bcl-2, Bax, caspase-3 and GAPDH (cat. no. KC-5G5; KangChen Biotech, Shanghai, China; dilution, 1:10,000) at 4°C overnight. The membranes were then incubated for 1 h at room temperature with the appropriate horseradish peroxidase conjugated-secondary antibodies. The blots were visualized using an enhanced chemiluminescence solution (EMD Millipore) and were exposed to X-ray film (Kodak, Tokyo, Japan). The density of the protein bands was analyzed using ImageJ 1.41 software (National Institutes of Health, Bethesda, MD, USA). The expression levels of the target proteins were normalized to those of GAPDH.
8
Western Blot Analysis of ALKBH5 and VEGFA
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RIPA (Beyotime Biotechnology, Haimen, China, #P0013B) supplemented with a protease inhibitor cocktail (Roche, Branford, CT, USA, #11697498001) and phosphatase inhibitor cocktail (Roche, Branford, CT, USA, #4906837001) was used for sample lysis. Proteins (15–30 µg) were loaded and separated by 10% SDS—PAGE and then transferred to polyvinylidene fluoride membranes. The membranes were incubated with the following primary antibodies at 4 °C overnight: ALKBH5 (1:1000; Abcam, Cambridge, UK), β-actin (1:5000; Cell Signaling Technology, Danvers, MA, USA) and VEGFA (1:1000, Abcam, Cambridge, UK).
The blots were further incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody (1:3000, Abmart, Shanghai, China) for 1 h at room temperature. Protein visualization was performed using an enhanced chemiluminescence solution (Merck Millipore, Darmstadt, Germany, #32106). The relative protein expression levels were analyzed by densitometry using ImageJ software. β-actin was used as a loading control.
The blots were further incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody (1:3000, Abmart, Shanghai, China) for 1 h at room temperature. Protein visualization was performed using an enhanced chemiluminescence solution (Merck Millipore, Darmstadt, Germany, #32106). The relative protein expression levels were analyzed by densitometry using ImageJ software. β-actin was used as a loading control.
9
Antiaris toxicaria Seed-derived Toxicarioside O Modulates Colorectal Cancer
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Human colorectal cancer cell lines HCT116 and SW480 cells purchased from the American Type Culture Collection were cultured in DMEM, supplemented with 10% fetal bovine serum (Biowest), 100 U/ml penicillin (Sigma), 100 μg/ml streptomycin (Sigma) at 37°C in an atmosphere containing 5% CO2. A BrdU Cell Proliferation Assay Kit was purchased from Abcam. An Annexin V-FITC/PI Apoptosis Detection Kit was purchased from KeyGEN Biotech. Enhanced chemiluminescence solution was from Merck Millipore. Toxicarioside O (TCO) was isolated and purified from the seeds of Antiaris toxicaria in our laboratory [4 ]. The purity of TCO was proved to be ≧95% by a chromatographic analysis. TCO was dissolved in DMSO and stored at −20°C for experiment use in this study. EX-527 was obtained from MedChemExpress. CQ and 3-MA were obtained from Sigma-Aldrich. The following antibodies were used in this study: phosphorylated and total forms of Akt, Phospho-p70S6 kinase, p70S6, phospho-4E-BP1, 4E-BP1, and Atg5 were purchased from Cell Signaling Technology; SQSTM1 and Beclin1 was from Santa Cruz Biotechnology; LC3 was from Sigma-Aldrich; PARP and SIRT1 was purchased from Abcam.
10
Western Blot Analysis of ADAM9 Protein
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Total proteins were extracted using the RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor (Solarbio, Beijing, China). The protein concentration was determined with the Bradford Protein Assay (Bio-Rad Laboratories, Inc.) according to the manufacturer’s instructions. An equal amount of protein (20 μg) was loaded into each well of the 15% SDS/PAGE and transferred on to the PVDF membranes (EMD Millipore, Billerica, MA, U.S.A.). The membrane was blocked with 5% non-fat milk for 1 h at room temperature (RT) followed by incubating with primary antibody against ADAM9 (1:1000; ab186833, Abcam, Shanghai, China) or GAPDH (1:2000; ab181602, Abcam, Shanghai, China) overnight at 4°C. After washing twice with TBST (0.1% Tween-20), the membrane was incubated with horseradish peroxidase-conjugated secondary antibody at RT for 1 h. The protein blots were detected using the enhanced chemiluminescence solution (EMD Millipore, Billerica, MA, U.S.A.) with the ChemDoc Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.).
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