Size-based separations of proteins by capillary electrophoresis using linear polyacrylamide as a sieving medium: model studies and analysis of cider proteins.
Electrophoretic conditions to separate sodium dodecyl sulfate (SDS)-protein complexes according to their relative molecular mass by capillary electrophoresis (CE) using linear polyacrylamide as a sieving matrix were examined. Five purified proteins with relative molecular masses between 14 400 and 66 200 Da were separated on a coated fused-silica capillary with an internal diameter of 100 microm and an effective length of 24 cm (total length, 32.5 cm). Benzoic acid was added to the solution of purified proteins as internal standard; beta-mercaptoethanol was also added as reducing agent. The running buffer composition was 0.05 M tris(hydroxymethyl)aminomethane (Tris), 0.035 M aspartic acid, 0.1% m/v SDS, 4% m/v acrylamide, the resulting pH being 8.0. The applied voltage was 7 kV (reversed voltage polarity) in order to avoid high current intensities. Under optimized conditions, the five proteins were separated in less than 15 min, with a % relative standard deviation (RSD) between 0.2 and 0.4 for migration times in the same day. Good efficiency (values between 150 000 and 40 000 N/m) and resolution (values between 2 and 2.8) were obtained. The inverse of relative migration times was found to correlate with the logarithm of their relative molecular mass. Finally, cider proteins were analyzed and their relative molecular masses were determined. These results were compared with those obtained by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
Department of Physical and Analytical Chemistry, University of Oviedo, Oviedo, Spain. dbg@sauron.quimica.uniovi.es
Domingo Blanco
DB
Gomis
Sara
S
Junco
Yoana
Y
Expósito
Ma Dolores
MD
Gutiérrez
D000180:Acrylic Resins; D002850:Chromatography, Gel; D019075:Electrophoresis, Capillary / Q000379:methods* / Q000592:standards; D004591:Electrophoresis, Polyacrylamide Gel; D027845:Malus / Q000737:chemistry; D011506:Proteins / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate; D000099166:Polyacrylamides
D000180:Acrylic Resins; D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D000099166:Polyacrylamides