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

作者：Nakano Keiichi; Tamura Shogo; Otuka Kohei; Niizeki Noriyasu; Shigemura Masahiko; Shimizu Chikara; Matsuno Kazuhiko; Kobayashi Seiichi; Moriyama Takanori^*

来源：Analytical Biochemistry, 2013, 438(2): 117-123.

DOI：10.1016/j.ab.2013.03.013

• Three-dimensional gel electrophoresis
• Monoclonal proteins
• Heterogeneity

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.

• 出版日期2013-7-15