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Two‐dimensional polyacrylamide gel electrophoresis of extracellular soybean pathogenesis‐related proteins using PhastSystem
Author(s) -
Roggero Piero,
Pennazio Sergio
Publication year - 1990
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150110117
Subject(s) - isoelectric focusing , polyacrylamide gel electrophoresis , chromatography , gel electrophoresis , coomassie brilliant blue , electrophoresis , sodium dodecyl sulfate , isoelectric point , chemistry , silver stain , polyacrylamide , glycoprotein , microbiology and biotechnology , biology , biochemistry , staining , enzyme , genetics
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 M r '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 r '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.