Assessment of small ligand-protein interactions by electrophoretic mobility shift assay using DNA-modified ligand as a sensing probe.
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 microM to 100 microM. 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.
Funabashi
Hisakage
H
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama , 226-8501, Japan.
Ubukata
Michito
M
Ebihara
Takashi
T
Aizawa
Masuo
M
Mie
Masayasu
M
Kobatake
Eiry
E
D000906:Antibodies; D001710:Biotin / Q000378:metabolism*; D002138:Calibration; D004247:DNA / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D024202:Electrophoretic Mobility Shift Assay; D008024:Ligands; D018625:Microscopy, Atomic Force; D015335:Molecular Probes / Q000378:metabolism*; D019809:Streptavidin / Q000378:metabolism*
D000906:Antibodies; D008024:Ligands; D015335:Molecular Probes; D001710:Biotin; D004247:DNA; D019809:Streptavidin