Basic analysis of 2DE images

First, two-dimensional gels must be digitized. In most cases, this is achieved by the use of a laser densitometer, a CCD camera or a phosphor imager [32]. These pieces of equipment produce images of typically around 2000 x 2000 pixels or more, and a depth of 12 or 16 bits, thus providing a dynamic range of 4096, respectively 65 536 grey levels.

Once loaded into the software, spot detection and quantitation is performed automatically, producing a repository of all protein spots contained in the 2-DE images, as well as related quantitative data, such as the spots' optical densities, area and volume (integration of the optical density over the area). Relative measures of these values are also given, as for example the relative volume calculated as the absolute volume divided by the total volume of protein in the whole 2-DE gel. Relative values allow to partially compensate for variations in sample load or staining. Using such relative quantitative values provides better reproducibility of data. Automatic 2-DE image comparison is then performed on user-selected gels, by a pattern-matching algorithm. This creates a unique mapping of all the protein spots in all considered 2-DE gels to a reference gel. The latter can either be one of the original 2-DE images or a synthetically produced image containing all (or a selection of) the spots contained in the analyzed gels. Melanie 3 provides several means to manipulate and display the gels, such as gel stacking, various zoom modes, customisable grids, "flicking" back and forth between two gels, or a transparency mode to visually inspect the result of matching. This makes it easy to browse through the data extracted from the 2-DE images. Figure 4.5 shows some of the display and navigation facilities of Melanie 3. Eight gels have been loaded. Four are visible, four are hidden and their names shown on the top right status line of the screen. On the four visible gels, one spot has been selected, the corresponding spot being displayed in green on all four gels. The top left part of the image shows two zoomed in, stacked gels (ECOLI and 94-005). Their spots are displayed in transparency mode, vectors allowing to examine the paired spots. A pop-up

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Display and navigation facilities in Melanie. For details see text.

zoom window shows the front gel zoomed out, a rectangle indicating what part of the gel is currently displayed. The bottom left gel displays some annotations (protein names and accession numbers). A second zoom window shows the same area, but zoomed in three times. An information window (bottom right) displays selected data related to the cursor position (in the current setting: gel name, spot ID, grey level value, pI/Mr10) calculated from the reference gel, optical density in the spot centre and spot area). Finally the top right gel is the synthetic gel resulting from merging the other three. It is displayed with a pI/Mw grid. The current pi and Mw values are shown in the top right, respectively bottom left part of the gel (only the pI range is visible on this Figure: [4.89, 5.99]).

Continue reading here: Annotations and databases

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