Approximately 300-μg proteins from each pooled samples were used for 2-DGE analysis. 2-DGE was performed using the Immobilized pH gradient (IPG) gel strips (17cm, pI 3-10 NL) for IEF and 8-16% pre-cast gels for SDS-PAGE (Bio-Rad). After staining with Sypro Ruby, the gels were scanned and analyzed using the PDQuest program (Bio-Rad). The proteins spots showing over 1.5 fold changes were excised for in-gel tryptic digestion, and the resulting peptides were analyzed by LC-MS/MS (Eksigent Nano-LC & Thermo LTQ), as previously described [31 ]. Database searching was performed against the International Protein Index (IPI) human proteome database using the SEQUEST search engine (Thermo Scientific).
Pdquest program
Manufactured by Bio-Rad
PDQuest is a software program developed by Bio-Rad for the analysis of 2D gel electrophoresis data. It provides tools for image capture, spot detection, spot matching, and quantitative analysis of protein expression.
Automatically generated - may contain errors
Lab products found in correlation
Sequest search engine, by Thermo Fisher Scientific (1 mentions)
Nano lc, by AB Sciex (1 mentions)
Gps explorer software, by Thermo Fisher Scientific (1 mentions)
4700 proteomicsanalyzer tof tof, by Thermo Fisher Scientific (1 mentions)
Ipgphor 2 apparatus, by Cytiva (1 mentions)
Gs 800 scanner, by Bio-Rad (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Mascot program, by Matrix Science (1 mentions)
Jmp software version 8, by SAS Institute (1 mentions)
3 protocols using pdquest program
1
Comparative Proteomics Analysis of Pooled Samples
2
2D Gel Electrophoresis Proteome Analysis
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GUTK or DMSO treated HepG2 cells were harvested and the protein concentration was determined using BioRad Dc protein Assay. A 2-DE was performed as described previously [33 (link)]. Briefly, the samples containing 150 μg of protein were diluted in a rehydration buffer, loaded onto IPG strips and rehydrated with an IPGphor II apparatus (Amersham). The isoelectric focusing was carried out in a stepwise voltage increasing manner. The gels were visualized by silver staining and the raw images were captured by using a GS-800 scanner and QuantityOne program, and subsequently analyzed by the PDQuest program (version 8.0, BioRad). The differentially expressed proteins spots were manually excised. After enzymatic digestion, the peptide samples were analyzed using a 4700 Proteomics Analyzer (TOF/TOF™) (Applied Biosystems). A peptide mass mapping was performed using a MASCOT program (Matrix Science, London) against Swiss-Prot database with a GPS explorer software (Applied Biosystems).
3
Statistical Analysis of Physiological Data
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Statistical analyses of physiological data were performed using the JMP ® software version 8.0 (SAS Institute, Cary, NC USA). The analysis was carried out separately for each sampling time (see Fig. 1). The interaction between PVX-SPCP1 and the different SA doses was evaluated for each parameter measured. To separate means a Fisher's Least Significant Difference (LSD) was conducted.
Spot detection and gel analysis was first conducted with the PDQUEST program (Bio-Rad) and the second time, manually. Normalization was performed with the regression model LOESS [15] . Only the spots present on all of the gel replicates were used for statistical analysis. Ratios between two expressed conditions were calculated as the mean of three independent values for each spot from the 2D gel electrophoresis analysis ± standard error. Spots lacking quantitative signal or too high quantitative variation between replicates, or spots with mixed proteins, were not considered for further analysis.
Spot detection and gel analysis was first conducted with the PDQUEST program (Bio-Rad) and the second time, manually. Normalization was performed with the regression model LOESS [15] . Only the spots present on all of the gel replicates were used for statistical analysis. Ratios between two expressed conditions were calculated as the mean of three independent values for each spot from the 2D gel electrophoresis analysis ± standard error. Spots lacking quantitative signal or too high quantitative variation between replicates, or spots with mixed proteins, were not considered for further analysis.
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