Poster: Genomic & proteomic resources

Abs # 880: Enhanced Visualization of the Medicago truncatula Leaf Proteome Through the Use of Affinity Chromatography

Presenter:	Donnelly, Bryna E., bedonnelly@noble.org
Authors	Donnelly, Bryna E. (A)   Watson, Bonnie S. (A)   Elmer, Aaron M. (A)   Sumner, Lloyd W. (A)
Affiliations:	(A): The Samuel Roberts Noble Foundation

Plant proteomes are commonly studied via two-dimensional electrophoresis (2-DE) which is often criticized for its limited ability to visual low abundance proteins. This is primarily due to the limited dynamic range of most protein stains and the profusion of certain proteins. For example, rubisco and other photosynthetic proteins make up an inordinately high percentage of the protein in leaf extracts which restricts the visualization of lower abundance proteins. In an attempt to enhance visualization of lower abundance leaf proteins, we have evaluated various affinity chromatography methods to selectively remove rubisco and other proteins of higher abundance prior to 2DE. Medicago truncatula leaf proteins were extracted using Tris buffer, protein concentration was quantified, and extracts loaded onto affinity columns. Proteins passing through the affinity columns in the effluent were collected, concentrated, and quantified. Proteins bound to the column were eluted with a salt gradient (NaCl), concentrated, buffer exchanged to remove salts, and quantified. One-dimensional SDS-PAGE was used to evaluate the efficiency and selectivity of various affinity supports for the removal of high abundance proteins such as Rubisco. The two most efficient affinity columns were selected and evaluated further via 2-DE experiments to assess column impact on global protein profiles quantitatively and qualitatively. The impact of affinity chromatography fractionation of green tissues on proteomic applications will be discussed.