For complex protein mixtures, including cell lysates, containing thousands of proteins, 2-D gel electrophoresis separation (2D-PAGE) is the standard method used to separate and isolate individual proteins for analysis. In addition to being useful for identifying individual proteins, it is also used to monitor changes in the amount of a specific protein or proteins as an indication of the onset of cancer in tissue or how cancer growth is affected by environmental influences or by treatment methods. We will develop a capillary equivalent to the standard 2D-PAGE method. It will offer several advantages compared to 2 D-PAGE, including automation, quantitative measurement in individual proteins, dramatically decreased run time, and reproducible size and pI measurement for protein identification. The main innovation is a scheme that will couple capillary iso-electric focusing (CIEF) with multicapillary capillary-gel electrophoresis (CGE) to allow automated two-dimensional capillary-gel electrophoresis of protein samples to be performed. In Phase I, the coupling scheme will be developed and tested. In addition, the fluorescence detection chemistry using """"""""on-column"""""""" detection on the capillary array will be developed. This will be a significant advance for the analysis of whole cell samples for clinical and research studies of cancerous and pre-cancerous tissue.
Many clinical and research labs involved in cancer research as well as proteomics research would benefit from the type of instrument that we are proposing to develop. Clinical labs for routine analysis would be our primary target, although a second design with more user control and flexibility for research applications would probably be the initial market.
| Bernard, Richard; Loge, Gary (2009) Poly(2-ethyl-2-oxazoline) as a sieving matrix for SDS-CE. Electrophoresis 30:4059-62 |
