SBIR-STTR Award

This proposal is to develop a complete integrated and automated system for silver staining and scanning two-dimensional high resolution gels to both increasing sensitivity and resolving power, and of improving quantitation. This system is required for studies on changes in gene expression in cells and tissues in response to drugs, xenobiotics, injury, stress and disease. The number of variables in the over 50 variations of silver staining published are large, making optimization using manual procedures extremely costly and tedious. In addition, quantitation is now realized to require dynamic measurements on each spot during image development since the kinetics of image development differs for different proteins. We have developed a prototype robotic silver staining system and propose its completion together with the development of a novel system for repeatedly scanning each gel during development. This requires a novel gel transport system, and a combined scanner-development system including a scanner which can require images at short intervals. The completed systems is expected to process up to 40 gels per day. The software required to match successive images, calculate integrated absorbances as a function of time, and, by reference to calibration runs, determine the amount of each protein present, will also be written. PROPOSED COMMERCIAL APPLICATION

Potential Commercial Applications:
A completely automatic computerized system for optimizing silver 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.

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.