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Biophysical Journal 90:3300-3314 (2006)
© 2006 The Biophysical Society

Development of a Novel GFP-based Ratiometric Excitation and Emission pH Indicator for Intracellular Studies

Ranieri Bizzarri, Caterina Arcangeli, Daniele Arosio, Fernanda Ricci, Paolo Faraci, Francesco Cardarelli and Fabio Beltram

NEST CNR-INFM, Scuola Normale Superiore, Pisa, Italy

Correspondence: Address reprint requests to R. Bizzarri, National Enterprise for nanoScience and Nanotechnology (NEST), Scuola Normale Superiore, Piazza dei Cavalieri, 7, I-56126, Pisa, Italy. Tel. 39-050-509434; E-mail: r.bizzarri{at}sns.it.

We report on the development of the F64L/S65T/T203Y/L231H GFP mutant (E2GFP) as an effective ratiometric pH indicator for intracellular studies. E2GFP 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 E2GFP ideally suited for imaging setups equipped with the most widespread light sources and filter settings. We used E2GFP to determine the average intracellular pH (pHi) and spatial pHi maps in two different cell lines, CHO and U-2 OS, under physiological conditions. In CHO, we monitored the evolution of the pHi during mitosis. We also showed the possibility to target specific subcellular compartments such as nucleoli (by fusing E2GFP with the transactivator protein of HIV, (Tat) and nuclear promyelocytic leukemia bodies (by coexpression of promyelocytic leukemia protein).




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D. Arosio, G. Garau, F. Ricci, L. Marchetti, R. Bizzarri, R. Nifosi, and F. Beltram
Spectroscopic and Structural Study of Proton and Halide Ion Cooperative Binding to GFP
Biophys. J., July 1, 2007; 93(1): 232 - 244.
[Abstract] [Full Text] [PDF]


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