Troubleshooting image analysis in 2DE.

Abstract

The image analysis part of gel-based proteome research plays an important role in the overall success of the experiment. The main purpose of software-assisted 2DE gel analysis is to detect the protein spots, match them between gels within an experiment, and identify any differences in protein expression between sets of samples. Efficient analysis of protein expression relies on automated image processing techniques. There are several factors to consider in the choice of software product, as well as in the implementation of the analysis itself. Successful quantification of protein expression levels is largely dependent on the algorithms for spot matching, normalization, and background subtraction provided by the 2DE analysis software. In addition to generic protocols for image acquisition and subsequent 2DE image analysis (using Progenesis PG200), this chapter describes methods for quantitative and qualitative evaluation of the quality of the image analysis.