Fluorescence labeling of a cytokine with desthiobiotin-tagged fluorescent puromycin.
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.
Sakamoto
Akihiko
A
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Yamagishi
Mai
M
Watanabe
Takafumi
T
Aizawa
Youichi
Y
Kato
Takashi
T
Funatsu
Takashi
T
D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D002460:Cell Line; D005456:Fluorescent Dyes / Q000737:chemistry*; D051379:Mice; D011691:Puromycin / Q000737:chemistry*; D013926:Thrombopoietin / Q000737:chemistry / Q000378:metabolism*
D005456:Fluorescent Dyes; D011691:Puromycin; D001710:Biotin; C004749:desthiobiotin; D013926:Thrombopoietin