While based on studies performed during the previous NIH grant period, this proposal outlines research which focuses in a fundamentally different direction. The overall goal of the research outlined in this proposal is to explore enhanced methods for separation of small protein samples. Three focus areas are outlined to achieve this overall goal. The first focus area is the primary area of research and involves development of open-tubular capillary electrochromatography (OT-CEC) for separation of microliter quantities of proteins. In this first area, very robust polyelectrolyte multilayer (PEM) coatings generated from alternate rinses of cationic and anionic polymers will be used in the OT-CEC mode. The ionic polymers which will be initially used in this phase of our research will be based on molecular micelles similar to those developed during our previous funding period. Other anionic polymers will be added as the research develops. The cationic polymers will also be polymers (e.g. poly-lysine) similar to those used during our previous funding period. However, new cationic molecular micelles will also be synthesized and explored in combination with our anionic molecular micelles as new forms of PEM coatings. In addition, we will explore the utility of zwitterionic polymers for PEM coatings in OT-CEC. A critical component of this research is to understand the structure of our PEM coatings. Characterization of our PEM coatings will be done in collaboration with Dr. Paula Hammond of the department of chemical engineering of MIT. The second focus area of this proposed research is to explore the applicability of the OT-CEC technology developed during the first phase to microfluidic platforms for analyses of nanoliter volumes of proteins. This research will be done in collaboration with Dr. Laurie Locascio of the National Institute of Standards and Technology. The third phase of this research involves the use of molecular micelles to improve the separation of proteins using gel electrophoresis. As with the other focus areas, various strategies will be exploited to optimize separations for different classes of proteins. Finally, it is noted that this project is relevant to almost all areas of biomedical research since proteins are ubiquitous. Thus, isolation, detection, and identification of small quantities of proteins as outlined in this proposal are important to almost all aspects of public health.
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