Decyder 2d difference analysis software

Gels were scanned with the Ettan DIGE Imager (software 1.0; GE Healthcare) and generated gel image triplets (Cy2, Cy3, and Cy5) comprising the CyDye-labeled proteins. Quantitative analysis was carried out with the DeCyder 2D difference analysis software (Version 6.5; GE Healthcare). Spot detection and matching was performed automatically with the DeCyder Batch processor. For spot detection, the estimated number of spots for each co-detection procedure was set to 2500. The best internal standard image (Cy2 labeled samples) based on the number of detected spots and overall similarity of the protein spot pattern with that of other gels was assigned as the “Master” and used as a template. The matching was checked manually in the biological variation analysis (BVA) module to ascertain the accuracy of the match process. Volume ratios were calculated for every spot on every gel by dividing the spot volume (Cy3 or Cy5), by the spot volume of the internal standard (Cy2), thereby correcting for inter-gel variations.

Gels were scanned with the Ettan DIGE Imager (software 1.0; GE Healthcare) and generated gel image triplets (Cy2, Cy3, and Cy5) comprising the CyDye-labeled proteins. Quantitative analysis was carried out with the DeCyder 2D difference analysis software (Version 7.0; GE Healthcare). Spot detection and matching was performed automatically with the DeCyder Batch processor. The gel-to-gel matching was also checked manually followed by statistical analysis of protein abundance change between samples in the biological variation analysis (BVA) module embedded in the DeCyder Software [37 (link), 79 (link), 80 ]. Spots of interest differentially expressed at least in two comparisons with p < 0.05, and spots differentially expressed in one comparison with p < 0.01 were further analyzed with MS (39 spots fulfilled the criteria, Fig. 1b).

Gels were scanned with the Ettan DIGE Imager (software 1.0; GE Healthcare) and gel image triplets (Cy2, Cy3 and Cy5) comprising the CyDye-labeled proteins were generated. Quantitative analysis was carried out with the DeCyder 2D difference analysis software (Version 7.0; GE Healthcare). Spot detection and matching was performed automatically with the DeCyder Batch processor. The gel-to-gel matching was verified manually followed by statistical analysis of protein abundance change between samples in the biological variation analysis (BVA) module embedded in the DeCyder Software [50 (link)–52 (link)]. Spots of interest with P < 0.05 were selected for identification. Spots with a p-value of P < 0.1 and with a change in expression ratio of at least +/- 30% (Average ratio, AR > |1.3|) in fluorescence level were also selected to increase the data set for the pathway analysis.