250 μg of protein sample was loaded into individual lanes on the focusing tray. The pre-wetted electrode wicks were overlaid with 11cm (pH 3–10) ReadyStrip IPG Strip (BioRad). The IPG strips were subjected to passive rehydration for 12 hours, before exposing to isoelectric focusing on Protean IEF Cell i11 (BioRad). The following conditions used in the 2DE run were: 250V for 20 minutes with linear ramp, 8,000V for 2.5 hours with linear ramp and 8,000V for 25,000 v-hours with rapid ramping. After isoelectric focusing, the IPG strips were equilibrated with pre-warmed DTT Equilibration Buffer I (BioRad) followed by iodoacetamide-Equilibration Buffer II (BioRad), for 10 minutes each on orbital shaker. The equilibrated IPG strips were further resolved by 12.5% Tris-HCl Criterion gel (11cm) (BioRad) at 200V for 65 minutes. After resolving the IPG strips, the gels were stained with InstantBlue (Expedeon) for 1 hour. Destaining was done thrice with water to remove excess stains on the gel. The gels were then scanned using GS-800 Calibrated Densitometer (BioRad). Detection of spots, spots matching and spot intensity analysis were performed using PDQuest 2-D Analysis Software (BioRad). Two biological repeats were obtained from two independent CHIKV-infection experiments.
Pdquest 2 d analysis software
Manufactured by Bio-Rad
Sourced in United States
PDQuest 2-D Analysis Software is a product offered by Bio-Rad for the analysis of two-dimensional (2-D) electrophoresis gels. The software's core function is to facilitate the detection, quantification, and comparison of protein spots within 2-D gel images.
Automatically generated - may contain errors
Lab products found in correlation
Gs 800 calibrated densitometer, by Bio-Rad (3 mentions)
Readystrip ipg strip, by Bio-Rad (3 mentions)
Imagescanner 3, by GE Healthcare (2 mentions)
Instantblue, by Abcam (1 mentions)
Mini protean tetra vertical electrophoresis system, by Bio-Rad (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Ipgphor ief system, by GE Healthcare (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Trans blot semi dry transfer cell, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Spss software 22, by IBM (1 mentions)
Immobiline drystrip, by GE Healthcare (1 mentions)
2 dimensional clean up kit, by GE Healthcare (1 mentions)
Rc dc protein assay kit, by Bio-Rad (1 mentions)
Versadoc imager, by Bio-Rad (1 mentions)
Protean ief cell, by Bio-Rad (1 mentions)
Acrylamide gel, by Bio-Rad (1 mentions)
Criterion vertical electrophoresis cell, by Bio-Rad (1 mentions)
Flamingo fluorescent gel stain, by Bio-Rad (1 mentions)
Any kd mini protean tgx precast gel, by Bio-Rad (1 mentions)
36 protocols using pdquest 2 d analysis software
1
2D Electrophoresis of CHIKV Proteins
2
Proteomic Analysis of Protein Samples
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Gels were incubated with fixation solution (12% acetic acid, 50% methanol) for 2 hours and treated with 50% ethanol for 20 min. After fixation, the gels were incubated with silver staining solution (0.2% silver nitrate) for 20 min to stain the gels and then developed in a 0.2% sodium carbonate solution. Stained gels were scanned with an Agfa ARCUS 1200 in an image format (Agfa-Gevaert, Mortsel, Belgium). PDQuest 2-D analysis software (Bio-Rad) was employed to analyze protein spots. Specific protein spots were cut from the gels, gel particles were digested with trypsin-containing buffer, and proteins were extracted for MALDI-TOF. Peptides of significance were investigated using the Voyager System DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA). MS-Fit and ProFound programs were applied to detect proteins, and protein sequences were identified from the SWISS-Prot and NCBI databases.
3
Proteomic Responses of Oysters to Ocean Acidification
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The proteomic responses of B2 oysters to ocean acidification and warming were assessed by two-dimensional gel electrophoresis (2DE). A total of three gels were run per treatment (representing each of the three replicate tanks per treatment). Proteins (150 μg in 125 μl of rehydration buffer containing 0.2% pharmalytes) were immobilised in pH linear gradient gel strips (7 cm, pH 4–7; ReadyStrip™ IPG Strips, Bio-Rad) by overnight passive rehydration. Isoelectric focusing (IEF) was performed using an IPGphor IEF System (GE Healthcare) at 100 V for 2 h, 250 V for 20 min, a gradient up to 5000 V for 2 h and then 5000 V for 2 h. Following IEF, gel strips were equilibrated for 20 min in equilibration buffer I (1% DTT, 75 mM of 1.5 M Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS, bromophenol blue) and then for 20 min in equilibration buffer II (2.5% iodoacetamide instead of 1% DTT in equilibration buffer I). Second dimension separation was conducted using 12% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-Rad) in a Mini-PROTEAN® Tetra Vertical Electrophoresis System (Bio-Rad). After electrophoresis, gels were stained with blue silver [41 (link)] and visualised using a ChemiDoc XRS+ (Bio-Rad). Quantitative image analysis of protein spots was performed by PDQuest 2-D Analysis Software (Bio-Rad).
4
Antigenic Protein Profiling of Trichinella spiralis
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The separated protein spots from the 2-DE gels were transformed onto PVDF membranes (Millipore, USA) using a Trans-blot semi-dry Transfer Cell TM (Bio-RAD, USA) for 90 min at 18 V. The membranes were blocked with 5% skimmed milk in PBS, pH 7.4 for 2 h at room temperature and then incubated with either T. spiralis infected swine pooled sera or infected mouse sera diluted 1:1 00 in PBS, pH7.4, with 0.05% Tween 20 (PBST). After 3 washes in PBST, the membranes were incubated with the horseradish peroxidase-conjugated rabbit anti-swine IgG or goat anti-mouse IgG (Sigma, USA). Reproducibility of the immune recognition was verified by repeating the immunoblot at least three times. The sera collected from normal swine or mice were used as controls. The PDQuest™ 2-D Analysis Software (Bio-RAD, USA) was used for matching and analysis of the antigenic protein spots on the 2-D gels.
5
Gel Imaging and Quantitative Analysis
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The stained gels were scanned using UMAX 2100XL-USB scanner (Bio-Rad, USA) at a resolution of 600 dpi. The gel images were saved in TIF format, and analyzed using PDQuest 2-D analysis software (Version 8.0, Bio-Rad, USA). Three gels with well-separated protein spots of each treatment were used to create “replicate groups” (Chandramouli et al., 2011 (link); Wang et al., 2014 (link)). To obtain the highest gel matching, manual editing was carried out after automated detection and matching. Control was used as reference gel. To account for quantitative variations, the data were normalized between samples using total intensity of protein spots based on the corresponding gel (Chandramouli et al., 2011 (link)). The normalized intensity of protein spots was averaged from 2-DE gels for three independent biological replicates. For statistical analysis, the SPSS software 22.0 (IBM SPSS, USA) was employed. Spots differing by 2-fold or greater volume between treatments were excised and used for protein identification (Li et al., 2015 (link)).
6
Two-Dimensional Gel Electrophoresis Analysis
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Images of the 2-DE gels were analysed using PDQuest 2-D Analysis Software (Bio-Rad, US). Average optical densities of spots were measured and compared using Student’s t-test at each time point. Each group had 6 gels per group corresponding to one gel per animal except the HFD and corresponding LFD control group at 2 weeks which had 5 gels per group as one gel from each group was removed due to low protein loading. Spots which showed significant differences between groups (p < 0.05) were manually cut from gels using a modified pipette tip.
7
Quantitative Proteomics of S-Nitrosylation
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2-DE spots’ pattern was analyzed with the PD Quest 2-D analysis software (Bio-Rad Laboratories). At least four independent experiments per condition were analyzed. An initial analysis was performed by using RFS images of each condition (Control and CysSNO-treated samples) and the same spots were matched in a single gel set and quantified. Spots were selected when the fluorescence signal was at least two-fold higher in CysSNO vs. control conditions in at least two replicates. For several spots, no signal was detected in the control conditions, or it was much lower than in CysSNO-treated samples. A quantitative analysis was performed in the remaining spots, calculating the mean CysSNO:Control ratio in each experiment, and significant differences were determined using an ANOVA test. Images of the total protein signal for each gel were matched in a separate gel set and quantified. The spots selected from the RFS analysis were manually matched to their corresponding total protein signal spots, and only those in which the amount of total protein remained constant were finally selected for identification.
8
Quantitative Proteomic Analysis Pipeline
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The gels were scanned using a Personal Densitometer system (GE healthcare, Piscataway, NJ, USA). The gel images were processed using the BioRad PDQuest 2-D analysis software (Ver. 8.0.1, Bio-Rad Laboratories, Inc., Hercules, CA, USA). The protein content was calculated based on the color intensity on the gels from two replicates.
The target protein spots were excised for the in-gel digestion. The digestion method was adopted as described by Xu et al.[29] (link). After digestion by trypsin at 37°C overnight, the resulting tryptic fragments were collected and vacuum dried for the MALDI-TOF/TOF analysis.
The target protein spots were excised for the in-gel digestion. The digestion method was adopted as described by Xu et al.[29] (link). After digestion by trypsin at 37°C overnight, the resulting tryptic fragments were collected and vacuum dried for the MALDI-TOF/TOF analysis.
9
Proteomic Analysis of LPS-Treated Cells
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The 38B9 cells that had been treated with LPS or LPS together with MK-D6 were lysed in lysis buffer. Each immunopellet was collected by immunoprecipitation and freeze-dried. Freeze-dried proteins were precipitated using the 2-dimensional (2-D) Clean-up Kit (GE Healthcare Life Sciences) before resuspension in rehydration buffer. The protein concentration was determined using the RC/DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Samples were loaded onto the strip holder, covered with an Immobiline Drystrip (GE Healthcare Life Sciences) and rehydrated passively at 20°C for 12 h. Isoelectric focusing was performed for a total of 20,000 V/h using a linear ramp protocol at room temperature. The strips were then equilibrated for 15 min each with buffer I and buffer II. The strips were loaded on top of the gels, and a 2-D run was performed at 70 V for 2 h. The gels were stained with Coomassie brilliant blue, and stained gels were analyzed using PDQuest™ 2-D analysis software (Bio-Rad).
10
Protein Visualization and Quantification
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The gels were individually stained with SyproRuby fluorescent stain (Bio-Rad Laboratories) according to the manufacturer's instructions. All gels were scanned at 100 mm resolution using the Molecular Imager FX™ at an excitation wavelength of 532 nm and an emission wavelength filter of 610 nm. Images produced from three independent extracts for each time point were converted into digital TIF files. Spot detection, pattern evaluation and normalization were performed using the PDQuest 2-D Analysis Software (version 7.2, Bio-Rad Laboratories). One gel from day 3 cell culture was set as master gel. Protein spots were automatically detected and visually checked for undetected or incorrectly detected spots and then matched to their corresponding spots in a digitized master gel. Intensity levels were normalized between gels by the total quantity in valid spots of gel images. In order to excise proteins or polypeptides from the gels they were visualized by silver staining.
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