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 language | English |
|---|---|
| Pages (from-to) | 238-242 |
| Number of pages | 5 |
| Journal | Journal of Bioscience and Bioengineering |
| Volume | 105 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2008 Mar |
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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 journal › Article
}
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
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
VL - 105
SP - 238
EP - 242
JO - Journal of Bioscience and Bioengineering
JF - Journal of Bioscience and Bioengineering
SN - 1389-1723
IS - 3
ER -