Fluorescence labeling of a cytokine with desthiobiotin-tagged fluorescent puromycin

Akihiko Sakamoto, Mai Yamagishi, Takafumi Watanabe, Youichi Aizawa, Takashi Kato, Takashi Funatsu

Research output: Contribution to journalArticle

Abstract

Fluorescence labeling of a cytokine at a specific site is required for observing cytokine-receptor interactions in living cells at the single-molecule level. Here, we demonstrated the C-terminus-specific fluorescence labeling of histidine-tagged thrombopoietin (TPO), a ligand for Mpl, with desthiobiotin-tagged fluorescent puromycin. Fluorescent TPO, purified by tandem affinity purification, stimulated the proliferation of Mpl-expressing cells. Within 10 min of its addition, fluorescent TPO was found to be diffusely distributed on the cell membranes of Mpl-expressing cells, and gradually accumulated to form fluorescent spots. This method is applicable for studying the spatial and temporal distributions of cytokines in individual living cells.

Original languageEnglish
Pages (from-to)238-242
Number of pages5
JournalJournal of Bioscience and Bioengineering
Volume105
Issue number3
DOIs
Publication statusPublished - 2008 Mar 1

    Fingerprint

Keywords

  • affinity tag
  • cytokine
  • desthiobiotin tag
  • fluorescence labeling method
  • fluorescence microscopy
  • puromycin
  • tandem affinity purification

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this