Proteomics-based approaches are gathering momentum, with complete characterization of an organism’s ‘proteome’ being routinely carried out in several laboratories worldwide in order to identify unique proteins in different species. One of the routine experiments of such proteomics approaches is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). 2D PAGE can be used for quick identification of various proteins or to study differential expression levels under different conditions. Manual characterization of the nearly 1000 proteins thus obtained cannot only be cumbersome but can also lead to errors. The availability of software for this purpose not only increases accuracy but also enables rapid screening of gels. Progenesis Software from GE Healthcare is designed specifically for this purpose.
The software requires an input of an 8-bit grayscale image of the gel, which can be obtained using any gel documentation system. The software can automatically generate a spot list, which can be used by the Ettan spot-picker (available from GE Healthcare) for spot picking. The Progenesis Software can compare a set of gels using any one user-defined gel as a reference. Initially, the gels have to be loaded into the software. The color, contrast and intensities of either individual gels or all the gels loaded for analysis can be changed according to your requirements. Next, the area to be compared is defined. Comparison is made in terms of the absence or presence of spots and in terms of differential spot intensities. Cut-offs can be user-defined or automated by loading user-saved or company-provided protocols. The software warps the gels digitally in order to adjust the spots between two similar 2D gels. The intensities of spots can also be adjusted so that the volumes of standard spots on two different gels are equalized. Gels can be calibrated using the software to enable error-free quantification of spot intensities. Background subtraction can also be carried out.
Very little manual intervention is required; this not only avoids errors but also offers advantages necessary for high-throughput screening. Initial automatic comparison of gels can take a few hours, as the software scans and documents the information of different spots on the gel under examination. Once the files are created for the different spots, manual comparison then becomes easy. Options for creating a 2D gel database using the software are also available. An HTML file can be made from a 2D gel, which helps in subsequent identification of similar protein spots on other 2D gels.
A three-dimensional image of the gel region of interest can also be obtained: This helps in obtaining a good perception of spot intensities and protein concentration. These 3D images can also be exported to Microsoft® PowerPoint slides for presentations. The software also offers the added advantage of providing an averaged gel of gels selected by the user. We use generally these averages for presentation of 2D data when a sample is not perfectly reproducible between runs.
The software comes with a descriptive manual and sample gel pictures for practice purposes. The downside of the software is that it is extremely expensive and requires a high-end computer to run it.