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A novel assay for the quantification of active transcription factors

Student thesis: Doctoral Thesis

Transcription factors are a family of DNA-binding proteins involved in the regulation of all cellular processes. One particular area of transcription factor function is the process of apoptosis, the deregulation of which is involved in cancer. The study of transcription factors may therefore provide a novel perspective on cancer aetiology, as well as insight into potential treatment opportunities. However, research into transcription factor activity has traditionally been hindered by technical limitations. Firstly, transcription factors being such potent molecules need only be present at a low concentration within a cell, thus requiring sensitive methods of detection, or a large cell sample for analysis. Secondly, their tertiary protein structures present problems for interactions with inhibitory pharmaceutical agents, limiting their clinical application.

The aim of this project was to develop a method to overcome these obstacles; a method for the quantification of active transcription factors, with the sensitivity for analysing endogenous transcription factors, and the high throughput adaptability as a screen for testing molecules altering a transcription factor’s DNA-binding activity. This method is based on the isolation of a synthetic oligonucleotide probe complexed with the transcription factor of interest, using Protein-A conjugated magnetic bead immunopurification. The synthetic oligonucleotide is subsequently quantified using quantitative Polymerase Chain Reaction. The complex of synthetic oligonucleotide, transcription factor and antibody was shown by the Electromobility supershift assay to form under a defined set of experimental conditions, and the synthetic oligonucleotide reliably detected by quantitative Polymerase Chain Reaction. Following optimisation, this method was able to quantify purified p53, with a sensitivity of 5 ng, However, in the scope of this project, performance was not sufficient for detection of endogenous p53 from LS174T cells. Further optimisations are required to reach this goal in order to compete with commercially available Enzyme Linked Immunosorbent Assay based methods.
Original languageEnglish
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Award dateMar 2011
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