TY - JOUR
T1 - Development of a highly sensitive three-dimensional gel electrophoresis method for characterization of monoclonal protein heterogeneity
AU - Nakano, Keiichi
AU - Tamura, Shogo
AU - Otuka, Kohei
AU - Niizeki, Noriyasu
AU - Shigemura, Masahiko
AU - Shimizu, Chikara
AU - Matsuno, Kazuhiko
AU - Kobayashi, Seiichi
AU - Moriyama, Takanori
N1 - Funding Information:
The authors thank Dr. Mitsufumi Nishio, Dr. Satoshi Hashino, and Dr. Katsuya Fujimoto (Hokkaido University Hospital, Sapporo, Japan) for serum sample collection. This study was partially supported by a Sapporo Biocluster “Bio-S” grant from the Knowledge Cluster Initiative of the Ministry of Education, Sports, Science, and Technology.
PY - 2013/7/15
Y1 - 2013/7/15
N2 - 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.
AB - 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.
KW - Heterogeneity
KW - Monoclonal proteins
KW - Three-dimensional gel electrophoresis
UR - http://www.scopus.com/inward/record.url?scp=84878068919&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84878068919&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2013.03.013
DO - 10.1016/j.ab.2013.03.013
M3 - Article
C2 - 23541520
AN - SCOPUS:84878068919
VL - 438
SP - 117
EP - 123
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -