Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser-induced fluorescence detection

Takashi Kaneta, Daisuke Yamamoto, Totaro Imasaka

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)

Abstract

The separation methods for proteins with high resolution and sensitivity are absolutely important in the field of biological sciences. Capillary sieving electrophoresis (CSE) is an excellent separation technique for DNA and proteins with high resolution, while LIF permits the most sensitive detection in CSE. Therefore, proteins have to be labeled with fluorescent or fluorogenic reagent to produce fluorescent derivatives. Both precolumn and oncolumn derivatization have been employed for the labeling of proteins in CSE. However, there is no report on the postcolumn derivatization due to the limitation in the use of a standard migration buffer, despite it being a promising method for sensitive detection of proteins. Here, we show a novel postcolumn derivatization method for protein separation by CSE, using a tertiary amine as a buffer component in the running buffer. Tris, which is commonly used as a base in CSE separation buffers, was substituted by tertiary amines, 2-(diethylamino)ethanol and triethanolamine. A buffer solution containing 2-(diethylamino)ethanol or triethanolamine can be used for the CSE separation followed by the postcolumn derivatization of proteins, since both reagents are unreactive toward a fluorogenic labeling reagent, naphthalene-2,3-dicarbaldehyde. Thus, LIF detection using the postcolumn derivatization permits significant reduction in the LOD (by a factor of 2.4-28) of proteins, compared with conventional absorbance detection.

Original languageEnglish
Pages (from-to)3780-3785
Number of pages6
JournalElectrophoresis
Volume30
Issue number21
DOIs
Publication statusPublished - Dec 2 2009
Externally publishedYes

Keywords

  • Capillary sieving electrophoresis
  • LIF
  • Postcolumn derivatization
  • Protein
  • Tertiary amine

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

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