In Vitro Transcription and Translation Coupled to Two-Dimensional Electrophoresis for Bacterial Proteome Analysis

Summary

The most popular approach for proteomic analysis is based on the combination of two-dimensional electrophoresis (2DE) and mass spectrometry. Although very effective, the method suffers from a number of limitations, the most serious one being the necessity of expensive and sophisticated instrumentation to be operated by skilled personnel. Here, we propose an alternative approach, which is particularly useful when one is interested to establish if a subset of proteins is present in a complex protein mixture derived from a sequenced organism. The method is based on amplification of the genes whose products are under investigation. The amplified genes are used in transcription and translation reactions and the derived radio-labeled proteins are separated by 2DE. The gel is autoradiographed and the autoradiograph is superimposed on the 2D gel (sample gel) from which the protein mixture from the organism has been separated. The matching between the autoradiographic spots and the protein spots of the sample gel allows immediate protein identification.