OCISBOxford Centre for Integrative Systems BiologyUniversity of OxfordNew Biochemistry Building
University of Oxford
South Parks Road
Oxford OX1 3QU

Tel: +44 (0)1865 613300
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Experimental Techniques

In order both to start to accurately model biological systems and to subsequently test those models we require as much detail as possible on the underlying biological parameters. These parameters can range from the exact molecular detail of the structures of the individual proteins, through the kinetics and dynamics of their interactions to the input:output characteristics of entire pathways and the effect on the physiology of the organism. We are using a variety of experimental techniques within the Centre to characterise these systems, some of which are highlighted below.

In vitro quantitation

Much work is being carried out using purified components of our target pathways. X-ray crystallography is being used to provide structural information on protein components both individually and in complexes. The specific kinetics of reactions, for example phosphotransfer rates, between individual proteins in signalling pathways is being calculated. Protein:protein interactions are being identified and the strength of their interaction quantified by techniques such as SPR and Mass spectrometry.

In vivo quantitation

Quantitative western blotting and proteomics are being used to determine the average copy number of proteins within cell populations. These data are being compared to the levels of transcription as measured by microarray chips. The use of fluorescent fusions to proteins of interest, combined with state of the art microscopy, is allowing the copy number, localisation, diffusion coefficients, interactions and turnover of protein components within complexes in individual cells to be quantified. Much of these data are being obtained with single molecule precision and over time to allow the dynamics of the systems of interest to be examined.

Phenotypic analysis

The roles of gene deletions, point mutations, protein depletion and overexpression on the physiology of the cells are being determined. The addition of carefully defined inputs into particular pathways combined with highly sensitive measurements of the outputs of the pathways is being undertaken to elucidate the structures and roles of the biological systems. These can be the roles of individual signal transduction pathways through to the interconnectivity of multiple pathways to bring about large scale phenotypic changes.

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