The densitometer scanner (BioRad, USA) was used to scan each two-DE at a 600 dpi resolution. Proteins detected in the digitalized images of each of the three replicates were analyzed using the PDQuest software (Bio-Rad, USA). All errors were corrected and the reliability of matches increased through spot by spot analysis of each gel. Besides spot detection, landmarks were aligned and identified, and matched spots were quantified. The DAPs showing reproducibility were marked using a software and/or numbered manually, as necessary. The proteins persistently visible in at least three replicates were considered for analysis. A correlation coefficient value of 0.8 was used for preparing match sets of each of the replicate gels. A change of at least 1.5-fold spot intensity at p < 0.05 and also any increase or decrease were considered.
Pdquest software
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
PDQuest software is a 2D gel electrophoresis analysis tool designed for the detection, quantitation, and identification of proteins. It provides tools for the analysis of protein expression patterns and the comparison of multiple gels.
Automatically generated - may contain errors
Lab products found in correlation
Gs 800 calibrated densitometer, by Bio-Rad (6 mentions)
Gs 800, by Bio-Rad (5 mentions)
Exquest spot cutter, by Bio-Rad (4 mentions)
Gs 710 imaging densitometer, by Bio-Rad (4 mentions)
Protean ief cell, by Bio-Rad (3 mentions)
Imagescanner 3, by GE Healthcare (3 mentions)
Ettan 3 system, by GE Healthcare (2 mentions)
Molecular imager fx, by Bio-Rad (2 mentions)
Gs 800 calibrated imaging densitometer, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Protein assay kit, by Bio-Rad (2 mentions)
Gs 900 calibrated densitometer, by Bio-Rad (2 mentions)
Gs 800 densitometer, by Bio-Rad (2 mentions)
Typhoon trio variable mode imager, by GE Healthcare (2 mentions)
Chemidoc mp, by Bio-Rad (2 mentions)
Densitometer scanner, by Bio-Rad (1 mentions)
Immobiline drystrips, by Bio-Rad (1 mentions)
Alphaimager hp, by Bio-Techne (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Eps 3500 xl power supply, by Cytiva (1 mentions)
111 protocols using pdquest software
1
Protein Expression Analysis by 2D-PAGE
2
Agronomic Parameter Analysis Protocol
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Agronomic parameter data are reported as mean ± SD of three independent experiments. Statistical analysis was performed by ANOVA followed by the Student-Newman-Keulus test, with the minimum level of significance being p < 0.05. Statistical significance of differences in protein spot densities from densitometric analysis of 2D-electrophoretic gels was assessed automatically by PD Quest software (Bio-Rad), using the Student’s t test (p < 0.05).
3
Isoelectric Focusing of Endosperm Proteins
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The isoelectric focusing (IEF) of the proteins from N04 endosperm at the three developmental stages was analyzed with three technical replicates. For each sample, equal amounts of total protein extract (1 mg) were assayed using 24 cm immobilized pH gradient (IPG) dry strips (Immobiline Dry-Strips, Bio-Rad, Hercules, CA, USA) with a linear pH gradient from 3 to 10. An active rehydration was performed overnight at 50 V. IEF was performed with an Ettan III system (GE Healthcare, USA) by increasing step by step from 50 to 10,000 V. Equilibration of the strips was performed immediately for 15 min with a buffer containing 0.375 M Tris-HCl (pH 8.8), 6 M urea, 20% glycerol, 4% SDS and 2% DTT, and followed for 15 min with a buffer containing 0.375 M Tris-HCl (pH 8.8), 6 M urea, 20% glycerol, 4% SDS and 2.5% iodoacetamide. Proteins from the IPG gel strips were separated using 12.5% SDS-PAGE gels. The gels were silver stained and scanned using a laser scanner (AlphaImager HP, Alpha Innotech, Santa Clara, CA, USA). Digital images of the gels were compared using Quantity One or PDQUEST software (Bio-Rad). The differentially expressed proteins (over two fold quantitative variations) were selected for further analyses. Differential spots in stained 2-dimensional gels were manually excised for protein digestion and LC-MS/MS analysis.
4
Differential Copper Binding Proteome
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Duplicate 2-DE gels were run three times for each treatment as described previously [14 (link)]. Briefly, for each replicate, 600 μg of copper-binding proteins were added to IPG dry strips during the rehydration step. After isoelectric focusing, gel strips were equilibrated in equilibration buffer. SDS-PAGE in the second dimension was then performed. Protein spots were visualized using a modified Coomassie brilliant blue staining method [16 (link)].
The analyses of gel images were carried out using the PDQuest software (Version 8.0; Bio-Rad). Student’s t-test was used for analysis of the difference between Cu-treated and control samples. Only spots that exhibited significant and reproducible changes between treatments were considered to be differentially expressed proteins.
The analyses of gel images were carried out using the PDQuest software (Version 8.0; Bio-Rad). Student’s t-test was used for analysis of the difference between Cu-treated and control samples. Only spots that exhibited significant and reproducible changes between treatments were considered to be differentially expressed proteins.
5
Proteomic Profiling by 2D Gel Electrophoresis
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For 2D gel electrophoresis, immobilized pH gradient (IPG) dry strips were equilibrated for 12-16 h with reswelling solution containing 7 M urea, 2% 3-[(3-cholamidopropy) dime-thyammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), and 1% pharmalyte. Next, 200 µg of the samples were loaded onto the strip; protein concentrations were determined by Bradford assay (Sigma-Aldrich). Isoelectric focusing (IEF) was carried out at 20℃ using a Multiphore II system (Amersham Biosciences, NJ, USA) and EPS 3500 XL power supply (Amersham Biosciences) according to the manufacturer's instructions. Prior to the second dimension, the focused IPG strips were reduced (1% DTT) and alkalized (2.5% iodoacetamide) in equilibration buffer (50 mM Tris-Cl, pH 6.8 containing 6 M urea, 2% SDS and 30% glycerol). SDS-PAGE was then performed with a Hoefer DALT 2D system (Amersham Biosciences) at 20℃. Two-dimensional gel electrophoresis (2-DE) gels were stained with Coomassie Brilliant Blue dye. Analysis of imaged spots was performed using PD-Quest software (version 7.0, BioRad). To determine the relative levels of protein in each individual spot, the spot volume of each protein was compared to that of a normalized protein volume.
6
Comparative 2D Gel Protein Analysis
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Gels were stained with colloidal Coomassie Brilliant Blue G-250 and documented using a GS-800 auto-calibrating imaging densitometer (Bio-Rad). Image analysis was performed using PDQuest software, version 8.0.1 (Bio-Rad). Comparative 2D data were derived from three biological replicates from each clot type (control clot and leprosy clot). The spots were quantified based on their relative ‘volume’: the amount of a protein spot was expressed as the sum of the intensities of all pixels composing that spot. To compensate for subtle differences in sample loading, gel staining and de-staining, the volume of each spot was normalized relative to the total density of valid spots present in the gel image. After automated detection and matching, manual editing was conducted.
7
Quantitative Proteomic Analysis by 2-DE
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Stained gels and developed films were scanned with the Molecular Imager ChemiDoc XRS System (Bio-Rad, USA) in transmission mode. All 2-DE gel images were captured, digitalized, and analyzed with PDQuest software (Bio-Rad) using tenfold over background as a minimum criterion for presence/absence. The analysis was re-evaluated by visual inspection. The spots present in all three biological replicates showing statistically significant, qualitative or quantitative differences between treatment (TR) and control (CK) were drawn after comparisons. On the basis of total quantity and intensity of valid spots in each 2D-gel, a normalization factor was generated. A normalized spot volume was determined for each protein spot. Data analysis was performed with Excel software (Microsoft). Error bars shown in figures represent the standard deviation values.
8
Statistical Analysis of Animal Weights
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The animal body weights were analyzed statistically using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc analysis performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, La Jolla Carlifonia USA, www.graphpad.com . The results were presented as means ± SEM. Statistical analysis for 2D gel analysis, the comparison between LF and HF groups was assessed using Student’s t-test and Boolean analysis sets by the PD Quest software (Bio-Rad, South Africa). The differences were considered statistically significant at p < 0.05.
9
2D Gel Electrophoresis Quantitation Protocol
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2D PAGE was performed essentially as described [47 ] by using both isoelectric focusing (IEF; using carrier ampholytes) and non-equilibrium pH gradient electrophoresis (NEPHGE). Proteins were visualized by autoradiography and/or silver staining according to published procedures [48 (link),49 (link)]. For quantitation, 2D gel autoradiographs were imaged using a Molecular Imager device (Bio-Rad Laboratories, Hercules, CA, USA) and images were analyzed using the PDQuest software (v.7.1; Bio-Rad Laboratories, Hercules, CA, USA). Gels were done in triplicate to minimize gel-to-gel variation. Only gels presenting well-focused spots and a limited amount of protein remaining at the origin were selected for quantitation. Protein levels were normalized to the levels of actin (IEF) and annexin II, which migrated in both IEF and NEPHGE gels.
10
Silver Staining for Protein Identification
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Silver staining was performed to detect and visualize protein spots. Gels were fixed with a fixation solution (12% acetic acid and 50% methanol) for 2 hr and stained with a sliver solution
(0.2% silver nitrate and 0.03% formaldehyde). After staining, gels were developed in a 0.2% sodium carbonate solution and scanned by Agfar ARCUS 1200™ (Agfar-Gevaert, Mortsel, Belgium).
Images of gels were analyzed by PDQuest software (Bio-Rad). Specific protein spots with intensity changes more than 2.5 times between vehicle + MCAO and quercetin + MCAO groups were selected
for protein identification. Selected protein spots were separated from the gels and silver stained gel particles were destained. After destaining, gel particle were digested by trypsin
containing buffer and peptides were collected for matrix-assisted laser desorption ionization time of flight (MALDI-TOF). Peptides were analyzed using a Voyager System DE™ STR
biospectrometry workstation (Applied Biosystem, Forster city, CA, U.S.A.) and protein was detected by MS-Fit (University of California, San Francisco, CA, U.S.A.) and ProFound programs.
Identification of protein sequences was performed by databases SWISS-PROT and National Center for Biotechnology Information (NCBI) databases.
(0.2% silver nitrate and 0.03% formaldehyde). After staining, gels were developed in a 0.2% sodium carbonate solution and scanned by Agfar ARCUS 1200™ (Agfar-Gevaert, Mortsel, Belgium).
Images of gels were analyzed by PDQuest software (Bio-Rad). Specific protein spots with intensity changes more than 2.5 times between vehicle + MCAO and quercetin + MCAO groups were selected
for protein identification. Selected protein spots were separated from the gels and silver stained gel particles were destained. After destaining, gel particle were digested by trypsin
containing buffer and peptides were collected for matrix-assisted laser desorption ionization time of flight (MALDI-TOF). Peptides were analyzed using a Voyager System DE™ STR
biospectrometry workstation (Applied Biosystem, Forster city, CA, U.S.A.) and protein was detected by MS-Fit (University of California, San Francisco, CA, U.S.A.) and ProFound programs.
Identification of protein sequences was performed by databases SWISS-PROT and National Center for Biotechnology Information (NCBI) databases.
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