Wheat cultivar discrimination by capillary electrophoresis of gliadins in isoelectric buffers.
A modified method is reported for screening of wheat cultivars: capillary zone electrophoresis of gliadins in isoelectric buffers. Previously published procedures recommended a 100 mM phosphate buffer, supplemented with 0.05% hydroxypropylmethylcellulose and 20% acetonitrile, in uncoated capillaries. Due to the very high conductivity of such a buffer (4.7 mmhos at 25 degrees C) high speed separations (10-12 min analysis time at 800 V/cm) could only be elicited in 20 microm internal diameter (ID) capillaries, at the expense of sensitivity. In the present report, we optimized the background electrolyte as follows: 40 mM aspartic acid (pH=pI=2.77) in the presence of 7 M urea and 0.5% short-chain hydroxyethylcellulose (Mn 27000 Da; apparent pH 3.9 in 7 M urea). As an alternative recipe, the same isoelectric buffer can be supplemented with a mixed organic solvent composed of 4 M urea and 20% acetonitrile (apparent pH 3.66). Due to the much lower conductivity (0.7 mmhos), separations can be carried out at 1000 V/cm in only 10 min, but in larger bore capillaries (50 microm ID), ensuring a five-times higher sensitivity. The gliadin patterns thus obtained are species-specific and allow easy identification of all cultivars tested of both durum and bread wheat. No adsorption of proteins to the silica wall seems to occur and high reproducibility in peak areas and transit times is obtained.
Capelli
L
L
Department of Agricultural and Industrial Biotechnologies, University of Verona, Italy.
Forlani
F
F
Perini
F
F
Guerrieri
N
N
Cerletti
P
P
Righetti
P G
PG
D002021:Buffers; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005903:Gliadin / Q000032:analysis*; D007525:Isoelectric Focusing; D012680:Sensitivity and Specificity; D014908:Triticum / Q000737:chemistry*
D002021:Buffers; D005903:Gliadin