|Obenland, David - Dave|
Submitted to: Postharvest Biology and Technology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/8/2003
Publication Date: 1/1/2004
Citation: Barraclough, D., Obenland, D.M., Laing, W., Carrol, T. 2003. A general method for tw-dimensional protein electrophoresis of fruit samples. Postharvest Biology and Technology 32(2004):175-181 Interpretive Summary: Proteins are important in determining the postharvest quality of fruit but their study is complicated by problems of low protein abundance and the presence of substances that interfere with protein extraction and separation. In this study, methodology was developed that enables a rapid and simple two-dimensional electrophoretic protein separation to be performed on a wide range of fruit tissues. These methods will help enhance the ability of researchers to study protein expression in fruit tissues and aid research directed toward the enhancement of fruit quality.
Technical Abstract: During experiments characterizing and identifying proteins from controlled atmosphere-stored apple and peach fruit, we optimized methods for the extraction and two-dimensional electrophoresis (2-DE) of fruit proteins, using commercially available immobilized pH gradient strips for the first dimension. The method is relatively rapid with minimal handling of small amounts of sample, and has been reproduced successfully for 2-DE of a variety of fruit and plant tissues in our labs. Critical factors for fruit tissues include using acetone precipitation following incubation in a lysis buffer, and a long iso-electric focussing time. We have observed no interference to focussing from such troublesome fruit components as soluble pectins, polyphenolics, or high-acidity fruit, using this protocol. In addition, we have used the method with no modification, for a range of fruit tissues including low protein sources (apple and peach flesh), high lipid material (avocado fruit flesh) and high acidity lemon tissue. As the method in our hands is straightforward and robust, we recommend the method for routine 2-DE separations of fruit samples.