Journal of Bioscience and Bioengineering 2008-03-01

Fluorescence labeling of a cytokine with desthiobiotin-tagged fluorescent puromycin.

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

Index: J. Biosci. Bioeng. 105 , 238-242, (2008)

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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.