In large 2-D DIGE proteomic studies with a large number of samples, it is essential to design the experimental setup to detect statistically significant protein changes under consideration of experimental variances. Herein are presented guidelines and general remarks on the extraction of protein expression data by following protein spots on their way from first spot synchronization, detection, quantification and statistical analysis until excision and identification. Further discussion addresses common difficulties, potential pitfalls and strategies for dealing with gel-to-gel discrepancies, labeling inefficiencies, and dye-and batch effects which might not be obvious to novices and even more experienced users of DIGE technology. Abbreviations. 2-D-DIGE, 2-D-difference gel electrophoresis; B[a]P, benzo[a]pyrene.