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Updated: Feb 4, 2026

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions
Published on: July 17, 2019
Evaluation of protein-ligand interactions using the luminescent interaction assay FlimPIA with streptavidin-biotin
Yuki Ohmuro-Matsuyama1, Takahiro Yamashita2, Keiko Gomi3
1Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, 4259-R1-18 Nagatsuta-cho, Midori-ku, Yokohama, 226-8503, Japan; The Japan Society for the Promotion of Science, 8 Ichiban-Cho, Chiyoda-ku, Tokyo, 102-8472, Japan; Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
Abstract:
Post-translational modifications, such as phosphorylation, are crucial in the regulation of protein-protein interactions and protein function in cell signaling. Here, we studied the interaction between the transactivation domain peptide of cancer suppressor protein p53 and its negative regulator Mdm2 using a novel protein-protein interaction assay, based on the modified FlimPIA using the streptavidin-biotin interaction to link the p53 peptide and the probe enzyme. We succeeded in detecting an attenuation in the affinity of p53 towards Mdm2 caused by the phosphorylation at Thr18. It showed that the targets, which are not easy to fuse with the FlimPIA probes, such as phosphorylated peptides can be used in this system. Also, the use of streptavidin nanobeads was found effective to get clearer signal, probably due to concentration of the detection system onto the bead surface. The system was further applied to the detection of FKBP-FRB interaction using biotinylated FKBP domain, which suggested another potential merit of this system that allows to avoid misfolding and steric hindrance often observed for the fusion protein approach.
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