Determining relative protein abundance using 2D-DIGE Figure 2 Determining relative protein abundance using 2D-DIGE. The relative protein abundance of a spot is defined as the normalized spot volume observed in the Cy3 or Cy5 channel (protein from a time point sample) divided by the normalized spot volume of the same spot measured in the Cy2 channel (protein reference pool) on the same gel. (a) gel images and three-dimensional 'landscape representation' of five protein spots identified as eukaryotic initiation factor (eIF)4A (or RNA helicase-1/ helicase 45; PF14_0655) of P. falciparum. The top panel ('Pool') shows a representative image (gel3; see Table 1) of the Cy2-labeled protein reference pool/internal standard, whereas the lower panels depict one typical image for each of the four time point (TP) samples (TP1: Cy3/gel3; TP2: Cy5/gel7; TP3: Cy3/gel4; TP4: Cy5/gel6). (b) Overlay image of the Cy3-labeled and Cy5-labeled eIF4A isoforms from TP1 (green) and TP2 (red) from gel1. (c) Summary of the quantitative DIGE **data** and the resulting relative protein abundance profiles for the five eIF4A isoforms derived from all eight gels. The table presents the corresponding relative standard deviations for each set of four abundance measurements (for a given spot and time point sample) as well as the median value of the four relative standard deviations for each spot. (d) Three-dimensional presentation of eIF5A (PFL0210c) isoform 1, which happened to be the spot exhibiting the greatest fold change in the entire analysis (15.1-fold increase in relative protein abundance between TP1 and TP4). 2D-DIGE, two-dimensional differential gel electrophoresis. in their expression pattern (for example, eIF5A). A detailed analysis and discussion of protein abundance and function is presented below.