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Assessment of small ligand-protein interactions by electrophoretic mobility shift assay using DNA-modified ligand as a sensing probe

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Abstract

The interaction between a small ligand and a protein were assessed by electrophoretic mobility shift assay. A sensing probe was created by modifying the model ligand, biotin, with DNA. The complex of DNA-modified ligand and anti-biotin antibody or streptavidin as a target protein was analyzed by agarose gel electrophoresis. The band corresponding to the DNA-modified ligand was shifted in the presence of the target protein, and the intensities of the shifted bands were decreased by adding increasing concentrations of free ligand ranging from 0.1 μM to 100 μM. From this calibration the concentration of ligand in the samples could be determined, allowing for evaluation of the interaction between a small ligand and its target.

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Acknowledgment

This work was supported in part by Grant-in Aid for Creative Scientific Research from Japan Society for the Promotion of Science (JSPS) and Grants-in-Aid from the Ministry of Education, Culture, Sports, and Technology (MEXT), Japan.

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Authors and Affiliations

  1. Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama , 226-8501, Japan

    Hisakage Funabashi, Michito Ubukata, Takashi Ebihara, Masuo Aizawa, Masayasu Mie & Eiry Kobatake

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  1. Hisakage Funabashi
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  2. Michito Ubukata
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  3. Takashi Ebihara
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  4. Masuo Aizawa
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  5. Masayasu Mie
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  6. Eiry Kobatake
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Correspondence to Eiry Kobatake.

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Funabashi, H., Ubukata, M., Ebihara, T. et al. Assessment of small ligand-protein interactions by electrophoretic mobility shift assay using DNA-modified ligand as a sensing probe. Biotechnol Lett 29, 785–789 (2007). https://doi.org/10.1007/s10529-006-9301-6

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  • DOI: https://doi.org/10.1007/s10529-006-9301-6

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