In Vitro Transcription and Translation Coupled to Two-Dimensional Electrophoresis for Bacterial Proteome Analysis
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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.
Book Title: In Vitro Transcription and Translation Protocols
Series: Methods in Molecular Biology | Volume: 375 | Pub. Date: May-03-2007 | Page Range: 183-209 | DOI: 10.1007/978-1-59745-388-2_10
Subject: Genetics/Genomics
Key Words: Bacterial proteomics - two-dimensional electrophoresis - in vitro transcription-translation -
Neisseria meningitidis B
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