Each CyDye (Cy3, Cy5 and Cy2) was resuspended in anhydrous N, N-dimethylformamide (DMF) to prepare a stock dye concentration of 1 mM. A working solution of 400 pmol of each CyDye was made by further dilution of the stock with DMF. Samples (test and control) were labeled with Cy3 and Cy5, while a mixture of equal amounts of all samples to be analyzed in the experiment, regarded as internal standard, was labeled with the third fluorescent dye; Cy2 according to the manufacturer's instructions (GE Healthcare). In brief, the pH of each sample was adjusted to 8.5 using 100 mM NaOH. 50 mg of each protein sample [malaria, controls (HC/FC) and internal standard] were separately labeled with 400 pmol of CyDyes. After addition of CyDyes, samples were incubated on ice for 30 in the dark. Labeling reaction was stopped by addition of 10 mM lysine followed by incubation on ice for additional 10 min. Dyeswapping was performed while labeling the test and control samples for eliminating any type of dye effects. After labeling, samples labeled with Cy3, Cy5 and Cy2 were mixed, diluted with the rehydration buffer and loaded on 18 cm, 4-7 pH IPG strips. Subsequent IEF and SDS-PAGE separation were performed following the same protocol as previously described in the 2DE section.