Nuclear proteins were separated by 2-DGE in order to establish a reference map. The images were analyzed by the PD Quest software. Computational analysis of the silver-nitrate-stained gels reproducibly revealed 312 different spots in the pH range 3-10. However, proteins in the basic pH range exhibited poor resolution. To make the reference map more comprehensive, we developed the proteome in the overlapping pH ranges 4-7 and 6-11 (Figures 22.2A and 22.2B). Indeed, reproducibility of high-resolution 2D patterns is an issue of concern for the basic pH range. Thus, sample cup-loading instead of in-gel rehydration was necessary. Consequently, 482 and 361 spots were detected at pH range of 4-7 and 6-11, respectively, that included the overlapping region. More than 600 exclusive spots were detected, out of which 572 spots survived the filtering process. A total of 160 spots with more than 30 quality score assigned by the software (based on the quality and quantity of the spots) were excised. Of these, 150 spots were identified with high confidence, and the corresponding protein spots are indicated on the gels (Figures 22.2A and 22.2B).
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