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Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies
被引:126
作者:

Bizzarri, R
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Scuola Normale Super Pisa, Natl Enterprise Nanosci & Nanotechnol NEST, CNR, INFM, I-56126 Pisa, Italy Scuola Normale Super Pisa, Natl Enterprise Nanosci & Nanotechnol NEST, CNR, INFM, I-56126 Pisa, Italy

Arcangeli, C
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Scuola Normale Super Pisa, Natl Enterprise Nanosci & Nanotechnol NEST, CNR, INFM, I-56126 Pisa, Italy Scuola Normale Super Pisa, Natl Enterprise Nanosci & Nanotechnol NEST, CNR, INFM, I-56126 Pisa, Italy

Arosio, D
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Scuola Normale Super Pisa, Natl Enterprise Nanosci & Nanotechnol NEST, CNR, INFM, I-56126 Pisa, Italy Scuola Normale Super Pisa, Natl Enterprise Nanosci & Nanotechnol NEST, CNR, INFM, I-56126 Pisa, Italy

Ricci, F
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Scuola Normale Super Pisa, Natl Enterprise Nanosci & Nanotechnol NEST, CNR, INFM, I-56126 Pisa, Italy Scuola Normale Super Pisa, Natl Enterprise Nanosci & Nanotechnol NEST, CNR, INFM, I-56126 Pisa, Italy

Faraci, P
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Scuola Normale Super Pisa, Natl Enterprise Nanosci & Nanotechnol NEST, CNR, INFM, I-56126 Pisa, Italy Scuola Normale Super Pisa, Natl Enterprise Nanosci & Nanotechnol NEST, CNR, INFM, I-56126 Pisa, Italy

Cardarelli, F
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Scuola Normale Super Pisa, Natl Enterprise Nanosci & Nanotechnol NEST, CNR, INFM, I-56126 Pisa, Italy Scuola Normale Super Pisa, Natl Enterprise Nanosci & Nanotechnol NEST, CNR, INFM, I-56126 Pisa, Italy

Beltram, F
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Scuola Normale Super Pisa, Natl Enterprise Nanosci & Nanotechnol NEST, CNR, INFM, I-56126 Pisa, Italy Scuola Normale Super Pisa, Natl Enterprise Nanosci & Nanotechnol NEST, CNR, INFM, I-56126 Pisa, Italy
机构:
[1] Scuola Normale Super Pisa, Natl Enterprise Nanosci & Nanotechnol NEST, CNR, INFM, I-56126 Pisa, Italy
关键词:
D O I:
10.1529/biophysj.105.074708
中图分类号:
Q6 [生物物理学];
学科分类号:
071011 ;
摘要:
We report on the development of the F64L/S65T/T203Y/L231H GFP mutant ( E 2 GFP) as an effective ratiometric pH indicator for intracellular studies. E 2 GFP shows two distinct spectral forms that are convertible upon pH changes both in excitation and in emission with pK close to 7.0. The excitation of the protein at 488 and 458 nm represents the best choice in terms of signal dynamic range and ratiometric deviation from the thermodynamic pK. This makes E-2 GFP ideally suited for imaging setups equipped with the most widespread light sources and filter settings. We used E(2)GFP to determine the average intracellular pH (pH(i)) and spatial pH(i) maps in two different cell lines, CHO and U-2 OS, under physiological conditions. In CHO, we monitored the evolution of the pH(i) during mitosis. We also showed the possibility to target specific subcellular compartments such as nucleoli ( by fusing E(2)GFP with the transactivator protein of HIV, ( Tat) and nuclear promyelocytic leukemia bodies ( by coexpression of promyelocytic leukemia protein).
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页码:3300 / 3314
页数:15
相关论文
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