Two-dimensional polyacrylamide gel electrophoresis of extracellular soybean pathogenesis-related proteins using PhastSystem.
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 PhastSystem 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 Mr'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 Mr's estimated. None of these acidic or basic soybean PR-Ps was a glycoprotein. PAGE with PhastSystem 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.
Roggero
P
P
Istituto di Fitovirologia Applicata, CNR, Torino, Italy.
Pennazio
S
S
D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005110:Extracellular Space / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D010935:Plant Diseases; D010940:Plant Proteins / Q000032:analysis*; D010942:Plant Viruses; D011933:Reagent Kits, Diagnostic; D013025:Glycine max / Q000032:analysis* / Q000382:microbiology
D006023:Glycoproteins; D010940:Plant Proteins; D011933:Reagent Kits, Diagnostic; C053376:pathogenesis-related proteins, plant