Development of a highly sensitive three-dimensional gel electrophoresis method for characterization of monoclonal protein heterogeneity

Keiichi Nakano, Shogo Tamura, Kohei Otuka, Noriyasu Niizeki, Masahiko Shigemura, Chikara Shimizu, Kazuhiko Matsuno, Seiichi Kobayashi, Takanori Moriyama*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Three-dimensional gel electrophoresis (3-DE), which combines agarose gel electrophoresis and isoelectric focusing/SDS-PAGE, was developed to characterize monoclonal proteins (M-proteins). However, the original 3-DE method has not been optimized and its specificity has not been demonstrated. The main goal of this study was to optimize the 3-DE procedure and then compare it with 2-DE. We developed a highly sensitive 3-DE method in which M-proteins are extracted from a first-dimension agarose gel, by diffusing into 150 mM NaCl, and the recovery of M-proteins was 90.6%. To validate the utility of the highly sensitive 3-DE, we compared it with the original 3-DE method. We found that highly sensitive 3-DE provided for greater M-protein recovery and was more effective in terms of detecting spots on SDS-PAGE gels than the original 3-DE. Moreover, highly sensitive 3-DE separates residual normal IgG from M-proteins, which could not be done by 2-DE. Applying the highly sensitive 3-DE to clinical samples, we found that the characteristics of M-proteins vary tremendously between individuals. We believe that our highly sensitive 3-DE method described here will prove useful in further studies of the heterogeneity of M-proteins.

Original languageEnglish (US)
Pages (from-to)117-123
Number of pages7
JournalAnalytical Biochemistry
Volume438
Issue number2
DOIs
StatePublished - Jul 15 2013

Keywords

  • Heterogeneity
  • Monoclonal proteins
  • Three-dimensional gel electrophoresis

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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