Epoxide Bonded Phases for Chromatography and Immunoprecipitation
Biomarker discovery is a difficult process. The goal of this work is to improve upon current state-of-the-art separation and extraction technology using functional epoxide surfaces. Specific attention is paid to immunoprecipitation and DNA separations. Immunoprecipitation is an excellent option when clean protein capture is required. Magnetic particles allow these experiments to be conducted in a high throughput fashion necessary for biomarker validation studies. However, commercial magnetic immunoprecipitation particles suffer from low particle magnetization, low capacity, and high nonspecific binding. By designing a new acid stable silica coated magnetic particle (Fe3O4@SiO 2) a two-fold increase in signal to nose, and almost three-fold increase in magnetization was observed over commercial magnetic particles. Recent advances in protein engineering has opened the door for new analytical techniques. With the discovery of transcription activator like effectors (TALE) protein specific DNA sequences can now be targeted. Development of a TALE chromatography column allowed the first ever separation of double stranded DNA via sequence recognition. This proof of concept work could allow a different method by which to study proteins that interact with double stranded DNA. The advantage of this method over a standard chromatin immunoprecipitation (ChIP) assay is that a specific sequence can be chosen without the need to transfect cells. Direct purification of specific DNA sequences would be possible without the need to transfect cells.
Wirth, Purdue University.
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