Image analysis workflow of a 2-DE gel electrophoresis experiment using Progenesis SameSpots (Nonlinear Dynamics). A. Sample preparation; B. 2-DE gel electrophoresis and gel staining; C. 2-DE image acquisition and image quality checked by the software automatically; D. Gel image alignment. A 2-DE gel image in the set of images for an experiment is manually set as the reference image (pink) by the user and then other 2-DE gel images (green) are aligned to the reference gel image one by one by a manual and/or automated way. E. After gel image alignment, the aligned images are grouped according to the experimental design. F. Extraction of proteins of interest. Spot volume normalization and calculation are performed by the software automatically.