Proteomics have come a long way since the mid 1990s, with many life-scientists adopting proteomic approaches to understand gene function. Whilst genomic information itself provides an excellent foundation for biomedical research, the complexity of a human cannot be explained by the genomics approach alone. Proteins are the ultimate effector of cell function, thus, studying the dynamic proteome complement of a cell at any given time is a better reflection of the immediate cell environment and its subsequent disease states. Proteins exert their roles in a living organism by interacting with other proteins, so it is believed that a protein's interactions and its functions are related. The study of protein interactions for biological discovery is now routinely carried out in high-throughput formats. Protein microarrays, in particular protein-protein interaction arrays, are valuable tools in protein functional studies, facilitating the unbiased systematic screening for potential interactors. This hypothesis- generating approach is essential in this post-genomic era to bridge the gap between genomics and proteomics, increasing the efficiency of biological research. This PhD describes the functional characterisation of Raf Kinase Inhibitor Protein (RKIP) using protein microarrays for the first time. Whilst the exact physiological role of RKIP remains unclear, RKIP has been associated with an increasing number of diseases, particularly in cancer. This microarray study provided an insight into RKIP’s role in the cell, with the identification of a broad spectrum of interactors not previously associated with its current known function. Findings from this study supported RKIP’s role as a metastatic suppressor, and its function as a scaffold protein. Whilst the advantages of protein microarrays for functional studies are clear, a number of limitations remain. The limitations associated with the current detection strategies in protein microarrays were addressed, with the application of a novel quantum dot-based detection strategy for the detection of protein interactions on microarrays. The performance of quantum dots was comparable to current Alexa-based detection strategies. Biological validation of the novel interactors are routinely carried out by immunoprecipitations (IP) and western blots, which introduces a bottle neck in the high throughput workflow. This thesis reports the development of an integrated co-IP / SILAC (stable isotope labelling by amino acids in culture) approach to compliment the rate of discovery made possible by microarrays.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:616329 |
| Date | January 2011 |
| Creators | Tan, Kit-Yee |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/2569/ |
Page generated in 0.002 seconds