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Abstract
A fully automated two-dimensional electrophoresis (2DE) system was employed for DNA aptamer selection against an unidentified protein in a mouse liver tissue extract as a model target. A 2DE-based systematic evolution of ligands by exponential enrichment (2DE-SELEX) was demonstrated for aptamer selection against a single protein spot that was separated on a nitrocellulose membrane. After four iterative 2DE-SELEX cycles, the oligonucleotide pool was sequenced and aptamer sequences were identified. A blotting assay showed that an identified aptamer with a stable stem–loop structure had specific binding activity against the target protein. The 2DE-SELEX was shown to be promising for the development of aptamers against unidentified proteins in complex samples for proteomic analysis and biomarker discovery.
Supplemental materials are available for this article. Go to the publisher's online edition of Analytical Letters to view the supplemental file.
Acknowledgments
Nasa Savory and Shinichi Goto contributed equally to this work.
This study was supported by the Industrial Technology Research Grant Project 2009 from the New Energy and Industrial Technology Development Organization of Japan (NEDO) and a grant from the Low-Carbon Research Network Japan (LCnet). K. Ikebukuro and N. Savory was supported by the Japan Society for the Promotion of Science (JSPS) Research Fellowships for Young Scientists DC1.
Notes
2DE-SELEX identified aptamers named MLT-417, MLT-419, and MLT-423, which were predicted to fold into relatively stable secondary structures, were used for binding analysis. MLT-429 was also selected for binding analysis as it was identified as two of 46 clones sequenced after 2DE-SELEX whereas the other sequences were unique.