Protein extraction and western blot analysis was performed as described previously (14 (link)). Protein was extracted by radioimmunoprecipitation assay lysis buffer (cat. no. C500005; Sangon Biotech Co., Ltd.) then quantified by bicinchoninic acid method. A total of 50 µg of protein was loaded per lane and separated by 4–12% SDS-PAGE and then transferred to a PVDF membrane. The membrane was blocked by 5% non-fat milk for 1 h at room temperature and subsequently incubated with primary antibodies for iNOS (1:1,000; Abcam, cat. nos. ab3523) and GAPDH (1:1,000; Santa Cruz Biotechnology Inc.; cat. nos. sc-32233) overnight at 4°C. Immunoreactivity was detected by a secondary horseradish peroxidase-conjugated IgG (1:5,000; Thermo Fisher Scientific, Inc.; cat. no. A32731) for 1 h at room temperature. Proteins were developed via enhanced ECL chemiluminescence reagent kit (cat. no. 32109; Thermo Fisher Scientific, Inc.) and visualized using Bio-rad Imaging systems (Bio-Rad Laboratories, Inc.) and quantified with Quantity One image software (v4.62, Bio-Rad Laboratories).
Quantity one image software
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Quantity One is an image analysis software developed by Bio-Rad for the capture, analysis, and quantification of 1D and 2D electrophoresis gels, Western blots, and other types of gel-based images. The software provides tools for image processing, data analysis, and report generation.
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Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Chemidox xrs system, by Bio-Rad (2 mentions)
Blocking buffer, by Ipsen (2 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Radioimmunoprecipitation assay (ripa), by Merck Group (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Thermo Fisher Scientific (2 mentions)
Ecl detection kit, by Merck Group (2 mentions)
Gel pro analyzer software, by Media Cybernetics (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated igg, by Thermo Fisher Scientific (1 mentions)
Imaging system, by Bio-Rad (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Anti opn, by Abcam (1 mentions)
Anti n cadherin, by Merck Group (1 mentions)
Anti axl, by Abcam (1 mentions)
Molecular imager chemidox xrs imaging system, by Bio-Rad (1 mentions)
Anti twist1, by Abcam (1 mentions)
23 protocols using quantity one image software
1
Protein Extraction and Western Blot Analysis
2
Evaluating EMT Markers by Western Blot
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The protein expression levels of OPN, Vimentin, E-cadherin, N-cadherin, Twist1, AXL and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were evaluated by Western blot. Total protein was extracted from cells by lysing the cells in RIPA buffer [50 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 1% NP-40, 0.25% Na-deoxydiolate, 1 mM EDTA] containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 mM Na3VO4, 1 Mm NaF). Protein samples was separated by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and transferred onto PVDF (polyvinylidene fluoride) membranes. After blocked with 5% non-fat milk/TBST, the membrane was incubated with the primary antibody. The following primary antibodies were used: anti-OPN (Abcam, Cambridge, UK), anti-Vimentin (Abcam), anti E-cadherin (Cell Signal Tech, Danvers, USA), anti-N-cadherin (Millipore, Massachusetts, USA), anti-Twist1 (Abcam), anti-Snail (Cell Signal Tech), anti-AXL (Abcam), or anti-GAPDH (Cell Signal Tech); and detected with enhanced chemiluminescence reagents (Thermo Fisher Scientific). Bands were acquired by Molecular Imager ChemiDox XRS+ Imaging System with Quantity One Image software (Bio-Rad Laboratories).
3
Western Blot Analysis of Protein Phosphorylation
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Total protein was extracted using lysis buffer containing a protease inhibitor cocktail. The nuclear and cytosolic proteins were previously prepared and stored at −80°C. For western blot analysis, 20 µg protein was separated by 12% SDS-PAGE, transferred to a polyvinylidene difluoride membrane (EMD Millipore), prior to being probed separately with goat polyclonal anti-phosphorylated (p)-PKC-β2 (cat. no. sc-11760; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), mouse monoclonal anti-PPAR-δ (cat. no. ab58137; 1:170; Abcam, Cambridge, UK) or mouse monoclonal anti-NF-κB inhibitor-interacting Ras-like protein 1 (NKIRAS1; cat. no. ab13666; 1:750; Abcam) primary antibodies. The primary antibodies were incubated overnight at 4°C and then incubated with horseradish peroxidase-conjugated secondary antibodies (cat. no. ab150115; 1:2,000; Abcam) 2 h at 37°C. The blots were then visualized using an Enhanced Chemiluminescence detection kit (Beyotime Institute of Biotechnology, Jiangsu, China). A Bio-Rad gel imaging system (Bio-Rad Laboratories) was used to capture images of the gels and the optical density values of the bands were determined using Quantity One image software (Bio-Rad Laboratories). The relative expression levels of target proteins were represented by the ratio of target protein bands to β-actin.
4
Tight Junction Protein Expression Analysis
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In this part, we applied the methods described in previous articles [19 (link)]. In brief, the total tissue proteins were collected for the determination of ZO-1, occludin, claudin-1, and claudin-2. By the way, equal amounts of total protein extracted from the ileal mucosa were respectively fractionated on 8%, 10%, and 12% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to polyvinylidene difluoride (PDVF) membrane (Millipore, Bedford, MA). Furthermore, after the membrane was blocked, the membranes were incubated with antibodies specific for ZO-1 (1:1000, Invitrogen), occludin (1:1000, Invitrogen), claudin-1 (1:1000, Invitrogen), claudin-2 (1:1000, Abcam), and β-actin (1:5000, Sigma) overnight at 4 °C. After washing with Tris-buffered saline Tween-20 (TBST), membranes were incubated with appropriate peroxidase-conjugated secondary antibodies (KPL, America) at room temperature for 60 min. The chemiluminescence signal was captured using a ChemiDox XRS system (Bio-Rad). The densities of the bands were quantified with Quantity One Image software (Bio-Rad).
5
Quantifying Liver Transcription Factors
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Liver tissues were snap-frozen at 1, 6 and 24 h after implantation and stored at −80°C. The tissues were homogenized and nuclear protein was extracted with a NE-PER Nuclear and Cytoplasmic kit (Pierce, USA). The levels of activator protein 1 (AP-1) and nuclear factor (NF)-κB were then measured by EMSA, as described elsewhere [30] . Detailed information of biotin-conjugated AP-1 and NF-κB consensus oligonucleotides is presented in Table 2 . The signal intensities were determined by enhanced chemiluminescence (ECL) reagents, and acquired with a Molecular Imager ChemiDox XRS+ Imaging System with Quantity One Image software (Bio-Rad, USA). ImageJ software was used to quantify the gray value.
6
Western Blot Analysis of FoxP3
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Transfected cells were plated in six-well plates for 48 h and then harvested. The total proteins were separated on 10% SDS–PAGE gels, followed by transferring to polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). After incubating with primary monoclonal antibody specific to FoxP3 (1 : 100, Abcam, Cambridge, UK) or GAPDH (1 : 2000, Beyotime, Shanghai, China), proteins were subsequently probed with horseradish peroxidase-conjugated goat anti-mouse IgG (1 : 4000, Santa Cruz, Dallas, TX, USA). Protein bands were visualised by enhanced chemiluminescence according to the manufacturer's instructions (Millipore). The relative intensity was determined using Quantity One Image software (Bio-Rad, Hercules, CA, USA). Each experiment was conducted three times, and data were averaged.
7
Western Blot Analysis of IDO Protein
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Cells were collected and lysed in RIPA (Millipore, Burlington, USA) supplemented with a cocktail (Sigma–Aldrich, St. Louis, MO, USA), 1 mmol/l PMSF (Thermo), and a phosphatase inhibitor (Thermo). For Western blots analysis, denatured samples containing the same amount of proteins (30 μg) were loaded onto the resolving gel (EpiZyme, Shanghai, China) for electrophoresis. Proteins were then transferred onto a nitrocellulose membrane (Whatman, Piscataway, NJ, USA). The membrane was blocked with blocking buffer (EpiZyme) and then incubated with primary antibodies at the following dilution IDO (1:200 dilution, LifeSpan Biosciences, Seattle, WA, USA) at 4°C overnight, respectively. The membrane was washed, incubated with the appropriate HRP-conjugated secondary antibody for 1 h, and then reacted with enhanced chemiluminescence substrate (Pierce, Rockford, IL, USA). The results were recorded by the Quantity One image software (Bio-Rad, Hercules, CA, USA) and relative intensity was calculated using the ImageJ software (NIH, USA).
8
Quantifying Temporal Protein Changes
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To quantify temporal changes in protein levels, Western blot analysis was performed as previous reported.14 (link) L4–L5 DRGs were removed quickly from deeply anesthetized rats and homogenized in an ice-cold lysis buffer containing 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mmol/L sodium orthovanadate, 1 mmol/L sodium fluoride, 1 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L phenylmethylsulfonyl fluoride, and 1% cocktail of protease inhibitors (Sigma-Aldrich). The lysates were then clarified by centrifugation at 12,000× g for 20 min at 4°C. Protein concentration was quantitated by the protein assay kit (Bio-Rad). Equal amount of samples was run on a 10% SDS-polyacrylamide gel electrophoresis and transferred to a polyphorylated difluoride membrane. The membranes were incubated with 5% nonfat dried milk for 60 min and then incubated with the primary antibodies at 4°C overnight. The membranes were incubated with an horseradish peroxidase-labeled secondary antibody and developed using the ECL detection kit (Millipore, Billerica, MA, USA). Protein expression was normalized against β-actin. Western blot bands were scanned and analyzed with the Quantity One image software (Bio-Rad).
9
Western Blot Analysis of Hepatic Inflammatory Markers
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The levels of TLR4, TNF-α, NF-κB p65, and phospho-NF-κB in liver tissue and hepatocytes were analyzed by western blotting. Proteins were extracted using RIPA buffer (Thermo, USA), according to the manufacturer’s instructions, and the protein concentrations were measured using a BCA protein assay kit (Thermo, USA). An equal amount of protein from each sample was subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Membranes were blocked with 5 % milk in 1 × Tris-buffered saline with 0.1 % Tween-20 (TBST) for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies. Membranes were washed twice for 10 min in 1 × TBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h. Membranes were then washed twice for 10 min in 1 × TBST. Proteins were visualized by ECL (Millipore), and blots were scanned using a Fluor-S MAX MultiImager System (Bio-Rad). The signal intensities were determined using Quantity One image software (Bio-Rad).
10
Western Blot Analysis of Placental Proteins
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We performed Western blot analysis as described previously34 (link). Briefly, the placenta was homogenized to obtain protein samples. Next, proteins (50 μg) were resolved by SDS-PAGE, transferred onto PVDF membrane, probed with a rabbit anti-mouse EST polyclonal antibody (1:200; Santa Cruz Biotechnology, CA, USA), and labeled with an HRP-labeled goat anti-rabbit IgG (1:5000; Millipore, Billerica, MA). Signal quantification was carried out using Quantity One image software (Bio-Rad).
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