Statistical analysis was performed, according to the procedures described in previously published papers [9 (link),18 (link),19 (link),20 (link),30 (link),31 (link),32 (link),33 (link),34 (link),35 (link)]. However, the statistical analysis procedures are briefly described as follows: “The images of 2D PAGE gels obtained from the renal proteome fractionation experiments (Groups C-60 and Hg-60) were analyzed with ImageMaster 2D Platinum 7.0 software (GE Healthcare). The analyses with ImageMaster 2D Platinum 7.0 software allowed us to obtain correlations between the images of gels (matching), through equivalence analysis between the protein spots in each 2D PAGE run. This also allows a comparison analysis among the protein spots in terms of distribution, volume, relative intensity, isoelectric point and molecular mass. The results of mercury determinations were expressed as M ± SD, and subjected to Student’s t test and F test to identify any significant differences, using SAS software (version 8). The significance level was set at 5%, that is, p < 0.05”. For protein expression data, statistical analysis was performed using the Protein Lynx Global Server software (version 2.5) (PLGS) of the nanoAcquity UPLC Xevo QTof MS equipment, as described in the previous section.
Imagemaster 2d platinum 7
Manufactured by GE Healthcare
Sourced in United States, Sweden, United Kingdom, Germany
The ImageMaster 2D Platinum 7.0 is a software application developed by GE Healthcare for the analysis and processing of 2D gel electrophoresis images. It provides tools for spot detection, quantification, and comparison of protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Imagescanner 3, by GE Healthcare (6 mentions)
Typhoon 9400 imager, by GE Healthcare (2 mentions)
Ettan ipgphor 3 system, by GE Healthcare (2 mentions)
Spss version 22, by IBM (1 mentions)
Protean ief cell, by Bio-Rad (1 mentions)
Mini protean cell, by Bio-Rad (1 mentions)
Focus fast silvertm stain, by G Biosciences (1 mentions)
Dharmafect 4 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Ettan ipgphor system, by GE Healthcare (1 mentions)
Typhoon 9200 scanner, by GE Healthcare (1 mentions)
Imagescanner, by GE Healthcare (1 mentions)
Immobilized ph gradient strip, by GE Healthcare (1 mentions)
2 d clean up kit, by GE Healthcare (1 mentions)
Ipg strip, by GE Healthcare (1 mentions)
Imagescanner 3, by RCI Labscan (1 mentions)
Coomassie blue g50, by Merck Group (1 mentions)
Imagescanner 2, by GE Healthcare (1 mentions)
Biotools, by Bruker (1 mentions)
Ettan dige imager, by GE Healthcare (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
51 protocols using imagemaster 2d platinum 7
1
Renal Proteome Fractionation and Statistical Analysis
2
Comparative Proteome Analysis of Mycobacterial Strains
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Image Master 2D Platinum 7.0 software (GE Healthcare) was used to measure the density of all protein spots and quantify the ANV for each protein spot. Statistical analysis (Student’s t-test, P ≤ 0.05) was performed using SPSS version 22.0 software (SPSS, Chicago, IL, USA) with a cut-off of 2-fold up- or downregulated to compare the ANV of each protein spot between two strains, i.e. PMD and PMD-R, PMD and UPK-R, and PMD-R and UPK-R strains.
3
Two-Dimensional Gel Electrophoresis of Salivary Proteins
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Salivary gland protein samples were immobilized on IPG strips (17 cm) of pH 3–10 (Linear, Bio-Rad) using a Protean IEF Cell (Bio-Rad) and kept for isoelectric focusing with a default cell temperature of 20°C and a maximum current of 50 mA/strip. Briefly, 300 μl sample in rehydration buffer were thawed and each of them were pipetted as a line along the edge of a channel. IPG strips were gently placed (gel side up) on to the sample and finally layered with 2–3 ml of mineral oil. The sample supernatants were rehydrated on IPG strips at 20°C overnight after which IEF was run in three steps: 1. 250V for 20 min Linear; 2. 10000V for 2.5 hrs Linear; 3. 10000V for 5–7 hrs and 40000 V-hrs in Rapid mode. After IEF run the strips were equilibrated in equilibration buffer I (6M urea, 0.375M Tris-HCl, pH 8.8, 2% SDS, 20% glycerol and 2% DTT) and equilibration buffer II (6M urea, 0.375M Tris-HCl, pH 8.8, 2% SDS, 20% glycerol and 2.5% iodoacetamide for 10 min. The second dimension was carried out on 12% SDS-PAGE on Mini Protean cell (Bio-Rad). The 2D gels were silver stained with FOCUS-FAST silverTM stain (G-Biosciences) after the run to visualize the spots. Stained gels of both the susceptible and resistant strains were scanned and analyzed using ImageMaster 2D Platinum 7.0 software (GE Healthcare Life Sciences).
4
Protein Abundance Analysis by 2D DIGE
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The Typhoon 9400 imager (GE Healthcare Life Sciences, NJ, USA) was used to scan for protein spots at wavelengths of 488/550 nm (cy2), 532/550 nm (cy3), and 633/550 nm (cy5), respectively. The 2D DIGE gel images were analyzed by the Image Master 2D platinum 7.0 software (GE Healthcare Life Sciences, NJ, USA) and the protein abundance changes for spot picking detection were calculated using cy3/cy2 and cy5/cy2 differential in-gel analysis ratios. Only spots with significantly different changes (P < 0.05) and in-gel ratios greater than 1.6 were excised from the silver stained gels for further analysis to identify specific proteins of interest.
5
Iturin A Modulates Akt in Breast Cancer Cells
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MDA-MB-231 and MCF-7 cells were plated in 70 mm Petri dishes in complete medium. Cells were starved for overnight with serum free medium. Cells were transiently transfected with 5 μg of pcDNA-Akt and empty vector with 7.5 μl of FuGENE® HD transfection reagent in 100 μl of Opti-MEM®I reduced serum media according to manufacturer’s protocol. After 24 h of transfection, the cells were treated with Iturin A for 24 h in serum free medium. siRNA-Akt was used to silence Akt expression. Briefly, cells were seeded in 70 mm Petri plates. After 70% confluency, cells were transfected with 14 mM siRNA-Akt in 6 μl DharmaFECT 4 Transfection Reagent (Thermo scientific) and 1 ml Opti-MEM®I reduced serum media according to manufacturer’s protocol. After 24 h of transfection, cells were treated with Iturin A for 24 h in serum free medium. After Iturin A treatment, both pcDNA-Akt and siRNA-Akt transfected cells were lysed with NP-40 lysis buffer and the protein expression profiles were checked by immunoblotting as discussed before. ImageMaster 2D Platinum 7.0 Software (GE Healthcare Life Sciences, NJ, USA) was used to plot comparative densitometric graph of blots. Iturin A treated transfected as well as control cells were also analyzed by flow cytometry for detection of apoptosis as per above described method35 (link).
6
Quantitative Analysis of Protein Levels
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GraphPad prism software was used to perform statistical analysis. Each experiment was carried out at least three times. The results with p-value<0.05 were considered significant. Image Master 2D Platinum 7.0 Software (GE Healthcare Life Sciences, NJ, USA) was used for densitometry analysis of blot. All data were represented as mean ± standard deviation (SD).
7
Proteomic Analysis of RKL9 and MRC2 Strains
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The gels were scanned using both the Fujifilm FLA-9000 Scanner, and HP Scanjet G4010 and the gel images were then analyzed and compared using the ImageMaster 2D Platinum 7.0 software (GE Healthcare, Sweden). The software performs gel alignment, spot averaging, and normalization between the control and test gel to determine the difference in expressed protein spots. Protein spots with P < 0.05 were considered differentially expressed. The fold change in protein expression spots was determined by comparing the ratio between the percentage volume of RKL9 and MRC2 strains.
8
Protein Extraction and 2D Electrophoresis
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Proteins were extracted using the method described by Hurkman and Tanaka (1986) (link) with certain modifications. The sample preparation procedure as well as 2D electrophoresis conditions used for separation are shown in Supplementary Table S1 , and were also described in details previously (Chmielewska et al., 2016 (link)). Plant material was extracted from two biological replications and two technical repetitions were prepared from each biological sample. Image Master 2D Platinum 7.0 software (IMP7, GE Healthcare, USA) was used for quantitative gel analysis. Protein spots’ relative volume (%vol.) was quantified. This parameter is relatively independent of sample loading and staining variation. In the case of roots, we could not proceed with 11 RILs due to insufficient material. For each genotype and tissue, four gels in control and four in drought conditions were analysed. The images of 2D electrophoretic gels were deposited in the Dryad Digital Repository (https://doi.org/10.5061/dryad.6st3v7r ; Rodziewicz et al., 2019 (link)).
9
Quantitative Protein Spot Analysis
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The gels were stained with Colloidal Coomassie Brilliant Blue (Neuhoff et al., 1985 (link)). ImageScanner III (GE Healthcare, USA) was used to scan the gels. Image Master 2D Platinum 7.0 software (GE Healthcare, USA) was used to quantitatively analyze the spots. The protein spots across the gels were matched automatically, however, manual edition was necessary to improve the analysis. Protein spots relative volume (% vol) was quantified. This parameter is relatively independent of variation due to protein loading and staining. Statistical significance of the relative change of accumulation of protein spots was determined using Student’s t- test.
10
Proteomic Analysis of Walnut Allergens
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Protein extracts from raw, dry-fried and boiled WNs were diluted with 50 and 300 µg/mL rehydration buffer (8 M urea, 2% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 3–10 NL 0.5% immobilized pH gradient [IPG] buffer, 0.002% bromophenol blue, and 50 mM dithiothreitol). The first-dimension separation was carried out on an Ettan IPGphor system (GE Healthcare, Chicago, IL, USA) in accordance with the manufacturer's instructions. The samples were loaded on 7- and 13- cm IPG strips, with pH 3–10 NL (GE Healthcare). Second-dimension separation was performed on 10% and 12% acrylamide gels, and colloidal Coomassie brilliant blue staining or electroblotting used for visualization. After electroblotting, the membranes were blocked using a similar procedure to that described above and incubated with 25 mL of the serum pool from WN sensitized subjects at a dilution of 1:3. Protein spots were visualized and analyzed using Typhoon 7000 FLA series and ImageMaster 2D Platinum 7.0 software (GE Healthcare).
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