Analysis of proteins in biological samples by capillary sieving electrophoresis with postcolumn derivatization/laser-induced fluorescence detection

Takashi Kaneta, Takehito Ogura, Totaro Imasaka

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Previously, we have demonstrated postcolumn derivatization of proteins separated by capillary sieving electrophoresis (CSE), in which naphthalene-2,3-dicarbaldehyde was employed as a fluorogenic labeling reagent. Standard proteins separated by CSE were reacted with naphthalene-2,3-dicarbaldehyde in the presence of 2-mercaptoethanol (2-ME) which plays a role of a reducing agent in the derivatization reaction. To improve the sensitivity, we attempted the use of ethanethiol instead of 2-ME. Ethanethiol showed 1.4- to 4.5-fold lower limits of detection for proteins than 2-ME. Furthermore, we found that 8-aminopyrene-1,3,6-trisulfonate (APTS) is a good marker for relative electrophoretic mobilities of proteins in CSE. Since APTS is a fluorescent and trivalent anion, it generates strong fluorescence and migrates faster than any of the proteins. Therefore, we employed APTS as a marker to obtain the relative electrophoretic mobilities of proteins. The present method was applied to the analyses of proteins in biological samples. Human Ewing's family tumor cell line 'RDES' was used as a sample. The cultured cells were lysed with a buffer containing Tris-HCl, NaCl, sodium dodecyl sulfate, and 2-ME. After denaturation, the lysate was directly introduced into the capillary. Several peaks, which would correspond to proteins with molecular mass ranging from 10 to 93kDa, were found in the cell lysate. In addition, we measured a milk sample by the CSE with postcolumn derivatization. The electropherogram showed five major peaks which corresponded to α-lactalbumin, β-lactoglobulin, κ-casein, bovine serum albumin, and mixture of α- and β-casein.

Original languageEnglish
Pages (from-to)1061-1067
Number of pages7
JournalELECTROPHORESIS
Volume32
Issue number9
DOIs
Publication statusPublished - Apr 1 2011
Externally publishedYes

Keywords

  • 8-Aminopyrene-1,3,6-trisulfonate
  • Capillary sieving electrophoresis
  • Laser-induced fluorescence
  • Postcolumn derivatization
  • Protein

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

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