Two-dimensional difference gel electrophoresis (2D DIGE) is a modified form of 2D electrophoresis (2DE) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as 'spots' with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. With the appropriate imaging system, DIGE is capable of reliably detecting as little as 0.5 fmol of protein, and protein differences down to +/- 15%, over a >10,000-fold protein concentration range. DIGE combined with digital image analysis therefore greatly improves the statistical assessment of proteome variation. Here we describe a protocol for conducting DIGE experiments, which takes 2-3 d to complete.

Ventral furrow formation is a key morphogenetic event during Drosophila gastrulation that leads to the internalization of mesodermal precursors. While genetic analysis has revealed the genes involved in the specification of ventral furrow cells, few of the structural proteins that act as mediators of ventral cell behavior have been identified. A comparative proteomics approach employing difference gel electrophoresis was used to identify more than fifty proteins with altered abundance levels or isoform changes in ventralized versus lateralized embryos. Curiously, the majority of protein differences between these embryos appeared well before gastrulation, only a few protein changes coincided with gastrulation,suggesting that the ventral cells are primed for cell shape change. Three proteasome subunits were found to differ between ventralized and lateralized embryos. RNAi knockdown of these proteasome subunits and time-dependent difference-proteins caused ventral furrow defects, validating the role of these proteins in ventral furrow morphogenesis.

Lights, Camera, Action! Systematic variation in 2-D difference gel electrophoresis images2-D Difference gel electrophoresis (DIGE) circumvents many of the problems associated with gel comparison via the traditional 2-DE approach. DIGE's accuracy and precision, however, is compromised by the existence of other significant sources of systematic variation, including that caused by the apparatus used for imaging proteins (location of the camera and lighting units, background material, imperfections within that material, etc.). Through a series of experiments, we estimate some of these factors, and account for their effect on the DIGE experimental data, thus providing improved estimates of the true relative protein intensities. The model presented here includes 2-DE images as a special case. Keywords IntroductionÜnlü et al.[1] developed a novel modified 2-DE technique called 2-D difference gel electrophoresis (DIGE), which circumvents many of the problems associated with comparison of separate gels. In 2-D DIGE, two different protein samples are pre-labeled with a pair of matched cyanine dyes which fluoresce at different wavelengths of light but have identical charge and almost identical mass, thus allowing two different samples to be run within one gel mixture in both dimensions. Because the two samples have been subjected to essentially the same environment during separation, the "between-gel" variation is significantly reduced; however, the "within-gel" variation that results from the imaging process is essentially unaffected [2,3]. This paper focuses on the systematic variation which occurs due to the DIGE data imaging process and the system used to image the proteins; for example, see Figs. 1 and 2.The purpose is to aid in the automatic detection of differential protein expression and modification. This problem's difficulty lies in developing a model that accurately represents the complexity of the experimental conditions. Only through extensive experimentation and deliberation have we successfully identified and modelled several nuisance effects influencing the overall experimental data. Section 2 describes the imaging apparatus and the sequence of steps performed in creating a protein image, thus helping to establish a statistical model that describes the effect caused by the system components. The general approach is widely applicable, while the specific model is appropriate for this particular type of imaging system. Materials and methods System descriptionThe fluorescence gel imager contains a cabinet and platform, and a scientific-grade CCD camera is situated and focused approximately five inches above the platform with a lamp approximately three inches away from the camera on each side. There are several machines of this specific type, while other commercial fluorescent gel imagers exist (e.g. a laser scanner).The camera image area is approximately 35 mm635 mm, while a gel is approximately 140 mm6175 mm. Thus, to Abbreviations: DIGE, difference gel electrophoresis; MS, mean square; MSE, mean squared erro...