TY - JOUR
T1 - Fluorescence labeling of a cytokine with desthiobiotin-tagged fluorescent puromycin
AU - Sakamoto, Akihiko
AU - Yamagishi, Mai
AU - Watanabe, Takafumi
AU - Aizawa, Youichi
AU - Kato, Takashi
AU - Funatsu, Takashi
N1 - Funding Information:
The authors thank Dr. Tomofumi Santa for designing the fluorescent puromycin, Dr. Hiroshi Miyazaki and his colleagues (Pharmaceutical Research Laboratory of Kirin Brewery Co., Ltd.) for providing scientific and technical information, and other members of our laboratory for helpful discussions. This work was supported by Takeda Science Foundation, Grants-in-Aid for Specially Promoted Research and Scientific Research (A), and the 21st Century COE program from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
PY - 2008/3
Y1 - 2008/3
N2 - 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.
AB - 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.
KW - affinity tag
KW - cytokine
KW - desthiobiotin tag
KW - fluorescence labeling method
KW - fluorescence microscopy
KW - puromycin
KW - tandem affinity purification
UR - http://www.scopus.com/inward/record.url?scp=41549101940&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=41549101940&partnerID=8YFLogxK
U2 - 10.1263/jbb.105.238
DO - 10.1263/jbb.105.238
M3 - Article
C2 - 18397775
AN - SCOPUS:41549101940
SN - 1389-1723
VL - 105
SP - 238
EP - 242
JO - Journal of Bioscience and Bioengineering
JF - Journal of Bioscience and Bioengineering
IS - 3
ER -