2024-03-29T09:58:19Zhttps://eprints.lib.hokudai.ac.jp/dspace-oai/requestoai:eprints.lib.hokudai.ac.jp:2115/530402022-11-17T02:08:08Zhdl_2115_20043hdl_2115_137Development of a highly sensitive three-dimensional gel electrophoresis method for characterization of monoclonal protein heterogeneityNakano, KeiichiTamura, ShogoOtuka, KoheiNiizeki, NoriyasuShigemura, MasahikoShimizu, ChikaraMatsuno, KazuhikoKobayashi, SeiichiMoriyama, TakanoriThree-dimensional gel electrophoresisMonoclonal proteinsHeterogeneity492Three-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.Journal Articleapplication/pdfhttp://hdl.handle.net/2115/53040https://eprints.lib.hokudai.ac.jp/dspace/bitstream/2115/53040/3/Manuscript%20.pdf0003-2697Analytical Biochemistry43821171232013-07-15enginfo:pmid/23541520info:doi/10.1016/j.ab.2013.03.013author