AUTOMATION OF QUANTITATIVE 2D GEL STAINING

本建议是自动化银和荧光染色和扫描 双向电泳凝胶的目的是检测和 测量响应药物的蛋白质丰度的变化， 外源性物质、损伤、压力、疾病和实验变量。为 对人体或动物组织的研究，非放射性检测方法是 这需要适用于非常大量的2-D凝胶。银 染色仍在开发中， available.到目前为止，还没有人开发出自动化的银或 能够处理所需通量的荧光染色系统 用于扩展毒理学和药理学研究，用于表征 来自不同细胞类型和组织的蛋白质，以及用于常规临床 化学，尽管有几次认真的尝试。在第一阶段，我们开发了 一个自动化的计算机控制的原型系统， 摄像头和图像分析系统。我们也做了初步的内部- 荧光染色和银染色程序的比较，以及 研究结果表明，这两项措施最终都是必要的。这些结果 为全面开发提供坚实的实验基础 第二阶段的系统，以及旨在 鉴定使用质谱法解析的蛋白质。 拟定商业应用： 一种用于优化银的全自动计算机化系统， 2D凝胶的荧光染色将是开发的关键因素 高通量2D蛋白质分析系统的重要性， 蛋白质组学的发展。这将使大规模的研究成为可能， 分子解剖学药理学和毒理学

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.