A capillary electrophoretic method for isolation and characterization of grape xylem proteins
AbstractEuropean (Vitis vinifera) and American (Vitis labrusca) grape species succumb to a bacterial disease known as Pierce's Disease (PD). In contrast, muscadine grape genotypes (Vitis rotundifolia) are tolerant/resistant to PD. This is due to the unique biochemical composition of muscadine xylem. However, because of low protein concentration, conventional methods such as low-pressure chromatography and PAGE are unsuitable for grape xylem protein characterization. In addition, these procedures are tedious, time-consuming and require large amount of sample. This study reports a procedure for isolating and separating proteins from muscadine and bunch grape xylem tissue. The procedure consists of separation of xylem from cortex and phloem, removal of pigments and other gummy substances from xylem with ethanol: ethylacetate (2:1) and subsequent Capillary Electrophoretic (CE) analysis of xylem protein extracts to achieve desired resolution. Number of peaks, peak height and areas, retention time and baseline position were used to compare resolution and study the effect of sample and separation buffer. Xylem tissue proteins extracted with 0.05% sodium borate buffer (pH 8.3) and subjected to CE using 1.2% sodium borate (pH 8.3) as a separation buffer were found to yield most satisfactory resolution of grape xylem proteins. The data obtained by CE were consistent and reproducible, and hence, is well suited to obtain excellent resolution of xylem tissue protein for identifying differences in protein composition among the grape genotypes.
(African Journal of Biotechnology: 2003 2(3): 66-70)