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

    Fingerprint

    Thrombopoietin
    Puromycin
    Labeling
    Fluorescence
    Cytokines
    Cells
    Cell membranes
    Cytokine Receptors
    Purification
    Ligands
    Histidine
    Molecules
    Cell Membrane
    desthiobiotin

    Keywords

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

    ASJC Scopus subject areas

    • Biotechnology
    • Bioengineering

    Cite this

    Fluorescence labeling of a cytokine with desthiobiotin-tagged fluorescent puromycin. / Sakamoto, Akihiko; Yamagishi, Mai; Watanabe, Takafumi; Aizawa, Youichi; Kato, Takashi; Funatsu, Takashi.

    In: Journal of Bioscience and Bioengineering, Vol. 105, No. 3, 03.2008, p. 238-242.

    Research output: Contribution to journalArticle

    Sakamoto, Akihiko ; Yamagishi, Mai ; Watanabe, Takafumi ; Aizawa, Youichi ; Kato, Takashi ; Funatsu, Takashi. / Fluorescence labeling of a cytokine with desthiobiotin-tagged fluorescent puromycin. In: Journal of Bioscience and Bioengineering. 2008 ; Vol. 105, No. 3. pp. 238-242.
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