Gels were scanned with ImageScanner III and analyzed using ImageMaster 2D Platinum v7.0 software (GE Healthcare, Uppsala, Sweden). The analysis was performed as previously described [5 (link)].
Imagemaster 2d platinum v7
Manufactured by GE Healthcare
Sourced in Sweden
ImageMaster 2D Platinum v7.0 is a comprehensive software package for the analysis of 2D gel electrophoresis images. It provides tools for image acquisition, processing, and analysis to facilitate the study of protein expression and modifications.
Automatically generated - may contain errors
Lab products found in correlation
Imagescanner 3, by GE Healthcare (4 mentions)
Version 12, by MedCalc (2 mentions)
Concentrator plus apparatus, by Eppendorf (1 mentions)
Maldi micro mx, by Waters Corporation (1 mentions)
Proteosilver plus, by Merck Group (1 mentions)
Jmp software, by SAS Institute (1 mentions)
Prism v6, by GraphPad (1 mentions)
Medcalc v 12, by MedCalc (1 mentions)
Imagescanner 2, by GE Healthcare (1 mentions)
Greyscale marker, by Kodak (1 mentions)
12 protocols using imagemaster 2d platinum v7
1
2D Gel Image Scanning and Analysis
2
2D Gel Electrophoresis Protein Analysis
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All 2-D CBB stained gels were scanned with high resolution scanner (GE Image Scanner III). Gel images (mel format) were analyzed using ImageMaster 2-D Platinum V.7.0 software (GE Healthcare, UK). For optimal clarity, protein spots were detected by adjusting different parameters, i.e., smoothness, saliency and minimum area. Spots detected near the edges of the gel were deleted manually to avoid erroneous interpretation. 2-D gel match sets were grouped into classes according to the task of analysis. Subsequently, each of the identified spots were marked and numbered. The matches with ANOVA value <0.05 were considered to be significant for analysis and the total number of protein spots in each sample were documented. The data for the proteins extracted by Protocols 1–3 from different tissues were generated from technical triplicates.
3
Comparative Proteomic Analysis of High Altitude
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The stained gels were scanned using an ImageScanner III (GE Healthcare, Wauwatosa, WI, USA) and images were analyzed using ImageMaster 2-D Platinum v7.0 software (GE Healthcare, Wauwatosa, WI, USA), with three independent biological replicates for the 2300 or 3300 m samples. Spots were automatically detected and matched; mismatched and unmatched spots were artificially modified through manual editing. Spot densities were expressed as mean normalized volumes, and fold changes were calculated between 2300 and 3300 m. Only spots with intensity ratios over 1.5-fold were selected as differentially expressed proteins (DEPs) and the over-expressed proteins at 3300 m vs. 2300 m were subsequently identified.
4
Protein Identification via MALDI-TOF MS
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Immunoreactive protein spots were identified in 2-D PAGE gels by taking reference points of the blotted membrane after temporary staining with Ponceau S solution, scanning the images, and then using ImageMaster 2D Platinum v7.0 software (GE Healthcare) for spot matching. The spots of interest were manually excised from gels and subjected to destaining and tryptic digestion as described previously (Cacciotto et al., 2010) . Peptide mixtures were dried in a Concentrator Plus apparatus (Eppendorf) , resuspended in 0.2% formic acid and then mixed with an equal volume of a-cyano-4-hydroxycynnamic acid as matrix (10 mg/mL in acetonitrile/0.2% TFA) (70:30, v/v), applied to the metallic sample plate, and air dried. Mass spectra were recorded on a MALDI micro MX (Waters, Manchester, UK) equipped with a reflectron analyser and used in delayed extraction mode; raw data, reported as monoisotopic masses, were then introduced into in-house MASCOT peptide mass fingerprinting search program (Version 2.2, Matrix Science, Boston, MA) and used for protein identification as described previously (Cacciotto et al., 2010) . Search parameters were as follows: peptide tolerance 50 ppm, enzyme trypsin.
5
Comparative Proteomic Analysis of Gels
6
Proteomic Analysis of Rett Syndrome
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2-DE gel images were analyzed using ImageMaster 2D Platinum v7.0 software (GE Healthcare). Spot intensity was expressed as protein percentage volume (%V). Comparative analysis between healthy controls and RTT patients of differently expressed proteins were evaluated using either Mann-Whitney rank sum test or Kruskal-Wallis test. Statistical significance was indicated by a two-tailed P-value <0.05. Data were expressed as mean ± standard deviations (mean ± SD) from triplicate determinations obtained in five separate experiments. The MedCalc version 12.1.4 statistical software package (MedCalc Software, Mariakerke, Belgium) was used.
7
Quantitative Analysis of Protein Abundance
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Gels were scanned and evaluated using ImageMaster 2D Platinum v7.0 software (GE Healthcare). After spot detection, 2D gels (three gels from three independent biological samples) were aligned and matched, and normalized spot volumes were determined quantitatively. For each matched spot, the percent volume was calculated as the volume divided by the total volume of matched spots. Spots with variations in abundance were subjected to ANOVA, Tukey–Kramer HSD tests and contrast analysis (JMP software, SAS Institute, Cary, NC, USA) to assign spots that significantly varied (p < 0.05) in abundance for two factors: drought and cultivar, and their interactions (Table S2 ). An unpaired two-tailed Student’s t-test was used to assign significant variations in abundance (a given drought treatment vs. control) within analyzed cultivars (Section 4.10 ; Table S1 ). Proteins were subsequently identified by MS from spots that significantly varied in abundance.
8
Image Analysis of Rett Syndrome
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Images of gels were analyzed using ImageMaster 2D Platinum v7.0 software (GE Healthcare). The reference gel for each group (i.e., healthy controls, RTT, R306C, T158M, R168X, and large deletions in exons 3 and 4) was defined and used for the comparative analyses. The background was subtracted from all gels using the average-on-boundary method. Spot volume was expressed as a ratio of the total protein percentage volume (%V) detected from the entire gel to minimize differences between gels (gel normalization), for pooling them. Only spots appearing in all gels of the same group were matched with those of the reference gel.
9
Comparative 2D Gel Electrophoresis Analysis
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ImageMaster 2D Platinum v7.0 software (GE Healthcare, Uppsala, Sweden) was used to analyze each image of gels. The reference gel was defined and used for the comparative analyses. Statistical analysis for protein differently expressed in the groups was carried out using GraphPad Prism software and the MedCalc version 12.1.4 statistical software package (MedCalc Software, Mariakerke, Belgium) was used. We considered “differently expressed” those spots being unmatched or having a significantly different percentage volume (%V) Nonparametric Mann–Whitney rank sum test or Kruskal–Wallis analysis of variance were used, as appropriate, to test the differences between groups. A two-sided p < 0.05 was considered to indicate statistical significance.
10
Gel-Based Proteomics Analysis Methods
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Gel imaging was performed by using ImageMaster 2D Platinum v7.0 software (GE Healthcare). A reference gel for each group was defined for the comparative analyses. The background was subtracted from all gels using the average on-boundary method. Spot volume was expressed as a ratio of the percentage volume (%V) detected from the entire gel to minimize differences between samples (i.e., normalization). Unmatched spots or spots with significantly different %V were considered differently expressed. Data were expressed as mean ± SD/SEM or medians [95% CI for median] as appropriate. Statistical analysis of protein variations was performed using multiple t-test with a false discovery rate (q) of 0.05. Differences between groups were tested using Kruskal-Wallis test or one-way ANOVA, with Dunn's or Holm-Sidak's multiple-comparisons tests for post hoc analyses. A two-tailed P value of less than 0.05 was considered statistically significant. The statistical software GraphPad Prism v6.01 (GraphPad Software Inc., La Jolla, CA, USA) and MedCalc v12.1.4 software package (MedCalc Software, Mariakerke, Belgium) were used.
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