Development of glutathione-coupled cantilever for the single-molecule force measurement by scanning force microscopy |
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Authors: | Yoshimura Shige H Takahashi Hirohide Otsuka Shotaro Takeyasu Kunio |
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Institution: | Laboratory of Plasma Membrane and Nuclear Signaling, Graduate School of Biostudies, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan. yoshimura@lif.kyoto-u.ac.jp |
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Abstract: | The accuracy and the fidelity of a single-molecule force measurement largely rely on how the molecule of interest is attached to the solid substrate surface (bead, cantilever, cover glass and etc.). A site-specific attachment of a protein without affecting its structure and enzymatic function has been a major concern. Here, we established a glutathione-coupled cantilever to which any glutathione S-transferase (GST)-fused proteins can be attached in a desired direction. The rupture force between glutathione and GST was approximately 100 pN on average. By using this cantilever, we succeeded in measuring the interaction force between importin alpha and importin beta. |
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Keywords: | AFM atomic force microscope APTES 3-aminopropyltriethoxysilane SATP N-succinimidyl S-acetylthiopropionate PBS phosphate buffered saline NHS N-hydroxysuccinimide GST glutathione S-transferase DTT dithiothreitol |
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