Two-dimensional polyacrylamide gel electrophoresis of extracellular soybean pathogenesis-related proteins using PhastSystem

✍ Scribed by Piero Roggero; Sergio Pennazio

• Publisher: John Wiley and Sons
• Year: 1990
• Tongue: English
• Weight: 567 KB
• Volume: 11
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/elps.1150110117

✦ Synopsis

Acidic and basic pathogenesis-related proteins (PR-Ps) were extracted from the intercellular fluid (IF) of soybean leaves, locally infected with tobacco necrosis virus and showing necrotic local lesions. Proteins were detected by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using PhastS y stem and precast commercially available gels. Extracts from healthy leaves were run as controls. PR-Ps were first run under native PAGE conditions or isoelectric focusing (IEF), the gels stained with Coomassie Blue, then run under sodium dodecyl sulfate (SDS)-denaturing conditions and finally stained with silver. Ten major acidic PR-Ps were separated; their M:s were close to those found by conventional PAGE. Their isoelectric points ranged from 3.5 to 5.0. Ten basic PR-Ps were separated and their M:s estimated. None of these acidic or basic soybean PR-Ps was a glycoprotein. PAGE with Phast-System and precast gels gives reliable results, comparable with those from conventional 2D-PAGE, with simpler experimental procedures. By electrophoresing Coomassie-stained gels with SDS in the second dimension, we were able to control the first-dimensional separation and to avoid laborious protocols generally adopted with unstained gels.