Proteomics instrumentation development in densitometry, electrophoresis, and microplates

Proteomic studies are highly dependent on instrumentation. In this project, aspects of electrophoresis, densitometry and microplate technology have been developed. In the work on densitometry, the first notion was to develop a refined laser densitometer system. In view of the prohibitive cost of such a system, adapted flatbed scanners were developed instead to quantify electrophoresis gels with the aid of accurate optical density calibration using step wedges. Various aspects of illumination using light emitting diode and liquid crystal display backlighting were also investigated. In advancing the microplate technology, the use of microplate wells to function like capillaries for filling of samples in high throughput screening without precise instrumentation was introduced. The principle of light shielding and total internal reflection by light emitting diodes was also implemented for non imaging quantification of protein samples without optical filters. With the imaging quantification approach, the problematic effects of point spread function and liquid meniscus of small liquid volumes on standard microplates were shown. In that vein, the capability of our capillary wells microplate design to overcome this limitation was demonstrated. In another aspect of imaging quantification, an innovative capillary wells microplate approach that permitted side viewing, as compared to standard microplates which only allows top viewing, was implemented. With this approach, errors arising from bubbles during measurement could be revealed and pipetting errors detected during actual assay runs. In the effort related to electrophoresis, we developed a coverslip fluidic approach that potentially could be used for simple and cost effective capillary electrophoresis. Currently, this is done using capillary tubes which makes quantification possible only with specialized instrumentation.