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52 protocols using image lab software v5

1

Agarose Gel Electrophoresis for DNA Analysis

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Each sample was mixed 1:4 with loading buffer (25 mg bromophenol blue, 10 mL Tris EDTA (TE) buffer 1× pH 8.0, and 20 mL glycerol with pH value adjusted to 8.0) and 10 mL aliquots were transferred into a 0.75% (w/v) agarose gel prepared using TAE buffer supplemented with 0.03 mL/mL GreenSafe Premium. Electrophoresis was run for 1.25 h at 150 mV. Gels were analyzed using a molecular imager GelDOC XR+ (BioRad, Hercules, CA, USA) and the resulting image was processed using Image Lab Software v5.1 (BioRad, Hercules, CA, USA). The band area for each positive control was manually defined (band intensity) and then copied into each sample lane (maintaining the distance to the wells; with the decrease in band intensity considered to be a result of a reduction of the amount of DNA present. The results were given as the percentage of inhibition of the DNA band degradation (for the antioxidant assay) (see Equation (2)) or as a percentage of DNA band degradation (for the pro-oxidant assay) (see Equation (3))
Inhibition of DNA degradation(%) =Intensity (Sample)Intensity (DNA solution) × 100
DNA degradation = 100 Intensity (Sample)Intensity (DNA solution)× 100
where Intensity (Sample) is the intensity of each sample band, and Intensity (DNA solution) refers to the intensity of the intact DNA solution (positive control).
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2

Western Blot Analysis of Protein Expression

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Western blot analysis was performed as described previously by the authors' team (15 (link)). The following antibodies were used and incubated overnight at 4°C: Anti-ORMDL3 (1:2,000; ab107639; Abcam), anti-SPHK1 (1:1,000; ab71700; Abcam) and anti-Tubulin (1:2,000; ab179513; Abcam), anti-GAPDH (1:5,000; ab181602; Abcam), anti-Claudin-18 (1:2,000; 21126-1-AP; ProteinTech Group, Inc.), anti-E-cadherin (1:2,000; YT1454; ImmunoWay Biotechnology), anti-phospho-ERK1/2 (1:1,000; ab201015; Abcam; kindly provided by Dr Xiao-Fei, Jiang) and anti-ERK1/2 (1:1,000; ab17942; Abcam; kindly provided by Dr Xiao-Fei, Jiang). Goat anti-rabbit horseradish peroxidase-conjugated IgG (1:5,000; ab97051; Abcam) was used to detect antibody binding at room temperature for 2 h. Target proteins were visualized using a ChemiDoc™ MP Imaging system (Bio-Rad Laboratories, Inc.) and analyzed using ImageLab software V5.1 (Bio-Rad Laboratories, Inc.).
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3

Quantifying Tumor Volume Dynamics

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An analysis of covariance (ANCOVA) (p < 0.05) was performed to compare the tumour volume between the TC (n = 15), LD-DXR (n = 16) and HD-DXR (n = 16) groups. The western blot experiments were conducted with technical repeats of n = 2 and biological repeats of n = 8. Bio-Rad Image Lab™ software v5.1 was used for normalization of the protein specific intensities against total protein intensities. Results were analysed in GraphPad Prism v7.0 by performing a one-way analysis of variance (ANOVA) with Bonferonni post hoc test (p < 0.05). The mean values ± standard error of the mean was reported. The fluorescent-based immunohistochemistry experiments were conducted with biological repeats of n = 8. Five images per section were analysed in Image J software v1.52a. The following equation was used to calculate the corrected total cryosection fluorescence: mean of integrated density – (mean of area of selected cell x mean fluorescence of background readings).
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4

Western Blot Analysis of Protein Expression

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Cells lysates were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Immunoblots were blocked in 5% milk in PBS-T (PBS plus Tween20) and probed with the indicated primary antibodies overnight at 4°C. Immunoblots were washed in PBS-T before incubation with the corresponding secondary antibodies. Rinsed immunoblots were developed using a Western Lightning Plus-ECL chemiluminescence kit (Perkin-Elmer, catalog no. NEL103001EA) and imaged in a Chemidoc XRS+ instrument (Bio-Rad, CA, USA). Densitometric analysis was performed using ImageLab software v5.1 (Bio-Rad, CA, USA).
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5

Protein Expression Analysis in Lung Tissue

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Briefly, 20 mg lung tissue was added to RIPA Lysis Buffer (cat. no. P0013B; Beyotime Institute of Biotechnology). The tissues were homogenised and total protein was extracted. The protein content was measured using a bicinchoninic acid assay. The protein sample was subjected to 10% SDS-PAGE with 30 µg/well and then transferred onto a PVDF membrane, before and the membrane was blocked at room temperature for 2 h with 5% skimmed milk and washed with TBS with 100.1% Tween-20 (TBST) three times, 10 min per wash. The primary antibody was prepared according to the manufacturer's protocol and incubated at 4˚C for 12-18 h. The dilution of the α-SMA antibody used was 1:2,000, whilst the dilution of fibronectin, vimentin, collagen I, collagen III, TGF-β1 and Smad3 antibodies was 1:1,000. The membranes were washed again with TBST three times for 10 min. Subsequently, the membrane was incubated with an HRP-conjugated secondary antibody (cat. no. 3999S; Cell Signalling Technology, Inc.) at room temperature for 1 h. Next, the membranes were washed with TBST three times for 10 min. Signals were visualized using chemiluminescence reagent (cat. no. 170-5061; Bio-Rad Laboratories, Inc.). Densitometry analysis was performed using Image Lab software (v.5.1; Bio-Rad Laboratories, Inc.).
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6

Mast Cell Tryptase Protein Detection

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Protein in the mast cell supernatants (75 µg of total protein) was precipitated in 15% trichloroacetic acid at 4°C overnight. The pellet was washed twice in cold acetone (−20°C) and solubilized in 75 μL of Laemmli buffer protein supplemented with TCEP (Bio-Rad). Samples were then heated at 95°C for 5 min, clarified by centrifugation at 12,000 g for 5 min, and 5 µg of the solubilized sample was loaded into 4%–20% Mini-Protean TGX precast gels (Bio-Rad, GmbH). After electrophoresis, the proteins were blotted onto a nitrocellulose membrane using the Trans-Blot Transfer Turbo System (Bio-Rad). The membrane was incubated with primary anti-mast cell tryptase antibody (sc-32889, Santa Cruz Biotechnology) at 1:1,000 dilution overnight at 4°C. Subsequently, the membrane was labeled with the secondary HRP-conjugated antibody (1:10.000; P0448, Dako) for 1 h at room temperature. Bands were visualized with ECL Select Western Blot Detection Reagent (GE Healthcare Life Sciences) by chemiluminescence (Chemidoc XRS; Bio-Rad). The molecular weight of each band was determined with the Image Lab Software v5 (Bio-Rad).
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7

Protease Activity Profiling by ABP Labeling

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Recombinant proteases (100 ng) were labeled with 1 µM ABP in reaction buffer in a final volume of 40 µL for 30 min at 37°C. After the addition of 4X Laemmli buffer (Bio-Rad, GmbH) supplemented with Bond-Breaker Tris (2-CarboxyEthyl)Phosphine (TCEP) Solution (Thermo Scientific, United States), the samples were heated at 95°C for 5 min and loaded into 4%–20% Mini-Protean TGX precast gels (Bio-Rad, GmbH). After electrophoresis, the proteins were blotted onto nitrocellulose membranes using the Trans-Blot Transfer Turbo System (Bio-Rad). Membranes were incubated with NeutrAvidin-HRP (1:50,000), and bands were visualized with ECL Select Western Blot Detection Reagent (GE Healthcare Life Sciences) by chemiluminescence (Chemidoc XRS; Bio-Rad). The molecular weight of each band was determined with the Image Lab Software v5 (Bio-Rad).
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8

Proteomic Analysis of Protease Activity

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The reaction product was then precipitated in 15% trichloroacetic acid at 4 °C during 90 min. The pellet was washed twice in cold acetone (−20 °C) and solubilized in 20 μL of protein solving buffer with tris-(2-carboxyethyl)-phosphine hydrochloride (PSB-TCEP; Macherey-Nagel, GmbH). Samples were then heated at 95 °C for 5 min, clarified by centrifugation at 12000 × g for 5 min and the solubilized sample was loaded into 4–20% Mini-Protean TGX precast gels (Bio-Rad, GmbH). After electrophoresis, the proteins were blotted onto nitrocellulose membranes by using the Trans-Blot Transfer Turbo System (Bio-Rad). Membranes were incubated with streptavidin-HRP (Life Technologies), and bands were visualized with ECL Prime Western Blot Detection Reagent (GE Healthcare Life Sciences) and quantified by chemiluminescence yield (Chemidoc XRS; Bio-Rad). The molecular weight and intensity of each band was determined with the Image Lab Software v5 (Bio-Rad). The bands corresponding to active proteases were identified by their sensitivity to AEBSF. Additionally, the activity index of each protease-corresponding band was estimated by the calculation of a ratio between the volumetric densitometry of the fluorescent signal generated by untreated vs AEBSF-pretreated duplicates (−/+AEBSF). An activity index of 0 was given to cluster bands that were not detected in specific samples.
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9

Quantitative Protein Analysis after CADA Treatment

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After CADA treatment, cells were collected and lysed in Nonidet P-40 buffer (1%, supplemented with 50 mM Tris HCl, pH 8.0, 150 mM NaCl, protease inhibitor cocktail (Roche, Basel, Switzerland) and PMSF). Lysates were run on between 4 and 12% Criterion XT Bis-Tris gels in MES buffer (Bio-Rad, Hercules, CA, USA), transferred to PVDF or nitrocellulose membranes using the BioRad Trans-Blot Turbo transfer system (Bio-Rad, Hercules, CA, USA), blocked with 5% non-fat dried milk in TBST and incubated with a primary and secondary antibody. SuperSignal West Pico and Femto chemiluminescence reagent (Thermo Fisher scientific, Waltham, MA, USA) was used for detection with a ChemiDoc MP system (Bio-Rad, Hercules, CA, USA). Signal intensities were quantified with Image Lab software v5.0 (Bio-Rad, Hercules, CA, USA). Differences in protein concentration between each lane were compensated by normalisation to the clathrin heavy chain or β-actin signal. To compare the down-modulating activity of CADA, IC50 values were calculated with GraphPad Prism 8 software (San Diego, CA, USA) on a four-parameter concentration–response curve fitted to data from at least three replicate experiments with flow cytometry. The absolute IC50 value represented the compound concentration that resulted in the 50% reduction in the protein level.
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10

Immunoblotting protocol for protein analysis

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Immunoblotting analysis was performed as previously reported (Vanhulle et al., 2022 (link)). Cells were collected and lysed in ice-cold NP-40 lysis buffer (50 mM Tris-HCL (pH 8.0), 150 mM NaCl, and 1% Nonidet P-40) supplemented with cOmplete Protease Inhibitor (Roche) and PMSF Protease Inhibitor (100 mM in dry isopropanol, Thermo Fisher Scientific). Cell lysates were centrifuged at 17,000 g for 10 min at 4°C to pellet nuclei and debris. For SDS gel electrophoresis, supernatant samples were boiled in reducing 2x Laemmli sample buffer (120 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 100 mM dithiothreitol, and 0.02% bromophenol blue). Equal volumes of lysate were run on Criterion XT Bis-Tris gels (4–12%; Bio-Rad) at 170 V for 55 min using 1x XT-MES buffer (Bio-Rad), transferred to nitrocellulose membranes using the BioRad Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked for 1 h with 5% non-fat dried milk in TBS-T (20 mM Tris-HCL (pH 7.6), 137 mM NaCl, and 0.05% Tween-20). After overnight incubation with primary antibody at 4°C, membranes were washed and incubated for 1h with secondary antibody. β-actin was used as a loading control. SuperSignal West Pico and Femto chemiluminescence reagent (Thermo Fisher Scientific) was used for detection with a ChemiDoc MP system (Bio-Rad). Signal intensities were quantified with Image Lab software v5.0 (Bio-Rad).
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