AUTOMATION OF QUANTITATIVE 2D GEL STAINING

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This proposal is to automate silver and fluorescence staining and scanning of two-dimensional electrophoresis gels with the aim of detecting and measuring alterations in protein abundance in response to drugs, xenobiotics, injury, stress, disease and experimental variables. For studies on human or animal tissues, non-radioactive detection methods are required which are applicable to very large numbers of 2-D gels. Silver staining is still under development and little data on quantitation is available. To date no one has developed an automated silver or fluorescence staining system capable of handling the throughput required for extended toxicological and pharmacological studies, for characterizing proteins from different cell types and tissues, and for routine clinical chemistry despite several serious attempts. In Phase I we have developed an automated computer-controlled prototype system including a digital camera and image analysis system. We have also done preliminary inter- comparisons of fluorescence staining and silver staining procedures, and the results suggest that both may ultimately be required. These results provide a solid experimental basis for the development of a full scale system for in Phase II, and for additional proposed studies aimed at identifying proteins resolved using mass spectrometry. PROPOSED COMMERCIAL APPLICATION: A completely automatic computerized system for optimizing silver and fluorescence staining of 2D gels will be a key factor in the development of a high throughput 2D protein analysis systems essential for the development of proteomics. It will make possible large scale studies in molecular anatomy, pharmacology, and toxicology.