Determination of dye/protein ratios in a labeling reaction between a cyanine dye and bovine serum albumin by micellar electrokinetic chromatography using a diode laser-induced fluorescence detection

Peng Jing, Takashi Kaneta, Totaro Imasaka

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

The degree of labeling, i.e., dye/protein ratio (D/P) is important for characterizing properties of dye labeling with proteins. A method for the determination of this ratio between a fluorescent cyanine dye and bovine serum albumin (BSA), based on the separation of the labeling mixture using micellar electrokinetic chromatography with diode laser-induced fluorescence detection, is described. Two methods for the determination of D/P were examined in this study. In these methods, a hydrolysis product and impurities, which are usually unfavorable compounds that are best excluded for protein analysis, were utilized to determine the amounts of dye bound to BSA. One is a direct method in which a ratio of the peak area of BSA to the total peak area of all the products produced in the labeling reaction was used for determining the average number of dye molecules bound to a single BSA molecule. The other is an indirect determination, which is based on diminution of all peak areas related to the products except for the labeled BSA. These methods were directly compared by means of a spectrophotometric method. The experimental results show that the indirect method is both reliable and sensitive. Therefore, D/P values can be determined at trace levels using the indirect method.

Original languageEnglish
Pages (from-to)2465-2470
Number of pages6
JournalElectrophoresis
Volume23
Issue number15
DOIs
Publication statusPublished - Aug 2002
Externally publishedYes

Fingerprint

Semiconductor Lasers
Bovine Serum Albumin
Chromatography
Labeling
Semiconductor lasers
Coloring Agents
Fluorescence
Proteins
Molecules
Fluorescent Dyes
Hydrolysis
Impurities

Keywords

  • Bioconjugate chemistry
  • Diode laser
  • Dye characterization
  • Micellar electrokinetic chromatography

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

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abstract = "The degree of labeling, i.e., dye/protein ratio (D/P) is important for characterizing properties of dye labeling with proteins. A method for the determination of this ratio between a fluorescent cyanine dye and bovine serum albumin (BSA), based on the separation of the labeling mixture using micellar electrokinetic chromatography with diode laser-induced fluorescence detection, is described. Two methods for the determination of D/P were examined in this study. In these methods, a hydrolysis product and impurities, which are usually unfavorable compounds that are best excluded for protein analysis, were utilized to determine the amounts of dye bound to BSA. One is a direct method in which a ratio of the peak area of BSA to the total peak area of all the products produced in the labeling reaction was used for determining the average number of dye molecules bound to a single BSA molecule. The other is an indirect determination, which is based on diminution of all peak areas related to the products except for the labeled BSA. These methods were directly compared by means of a spectrophotometric method. The experimental results show that the indirect method is both reliable and sensitive. Therefore, D/P values can be determined at trace levels using the indirect method.",
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