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Methods in molecular biology (Clifton, N.J.)

Capillary electrophoresis of seed storage proteins: the separation and identification of microheterogeneous rice glutelin subunits.


PMID 23386349

Abstract

Glutelin, the major seed storage protein of rice (Oryza sativa L.), consists of multiple polymeric and monomeric subunits. Each subunit is composed of an α and a β polypeptide that are covalently linked by a disulfide bond. To analyze the microheterogeneous glutelin subunits using capillary electrophoresis (CE), the author identified the appropriate sample preparation procedures as well as optimal CE conditions. The glutelin was dissociated into its component α and β polypeptides by denaturation and reduction with low urea and 2-mercaptoethanol for a long incubation time at room temperature. The molecular species of the completely dissociated α and β polypeptides were identified and quantitatively analyzed by CE and SDS-PAGE using glutelin mutants. The measured CE migration times of the polypeptides correlated well with the calculated charge-to-size parameter values. Therefore, the rapid, simple, and precise separation and quantification of microheterogeneous proteins by CE required not only optimal CE conditions but also adequate protein pretreatment based on the molecular nature of the protein tested.