Fluorescence polarisation of green fluorescent protein (GFP). A strategy for improved wavelength discrimination for GFP determinations

Abstract

The fluorescence of green fluorescent protein (GFP), present both within whole yeast cells and in protein extracts from yeast cells, has been observed to be significantly polarised. Fluorescence polarisation is proposed as a useful technique to allow some discrimination between GFP fluorescence and that of other interfering species in cell or media matrices, which have fluorescence bands that overlap those of GFP, which should lead to improved resolution and limits of detection. The method has been tested by discriminating between the fluorescence of GFP in cell extracts and added fluorescein, both of which fluoresce brightly at the same wavelength. The flow-through instrumentation incorporating an argon-ion laser developed for this work is also described.

References

1. Green Fluorescent Proteins: Proteins, Properties, Applications and Protocols, ed. M. Chalfie and S. Kain, Wiley and Sons, 1st edn., 1998.
2. A. B. Cubitt, R. Heim, S. R. Adams, A. E. Boyd, L. A. Gross and R. Y. Tsien, Trends Biol. Sci., 1995, 20, 448.
3. H.-H. Gerdes and C. Kaether, FEBS Lett., 1996, 389, 44.
4. T. Misteli and D. L. Spector, Nat. Biotechnol., 1997, 15, 961.
5. F. Yang, L. G. Moss and G. N. Phillips, Jr., Nat. Biotechnol., 1996, 14, 1246.
6. R. Tombolini, A. Unge, M. E. Davey, F. J. deBruijn and J. K. Jansson, FEMS Microbiol. Ecology, 1997, 22, 17.
7. G. H. Patterson, S. M. Knobel, W. D. Sharif, S. R. Kain and D. W. Piston, Biophys. J., 1997, 73, 2782.
8. N. Billinton, M. G. Barker, C. E. Michel, A. W. Knight, N. J. Goddard, P. R. Fielden and R. M. Walmsley, Biosens. Bioelectron., 1998, 13, 831.
9. K. D. Niswender, S. M. Blackman, L. Rohde, M. A. Magnuson and D. W. Piston, J. Microsc., 1995, 180, 109.
10. M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward and D. C. Prasher, Science, 1994, 263, 802.
11. M. J. Zylka and B. J. Schnapp, Biotechniques, 1996, 21, 220.
12. M. Kneen, J. Farinas, Y. X. Li and A. S. Verkman, Biophys. J., 1998, 74, 1591.
13. R. Swaminathan, C. P. Hoang and A. S. Verkman, Biophys. J., 1997, 72, 1990.
14. B. P. Cormack, G. Bertram, M. Egerton, N. A. R. Gow, S. Falkow and A. J. P. Brown, Microbiology, 1997, 143, 303.
15. R. M. Walmsley, N. Billinton and W.-D. Heyer, Yeast, 1997, 13, 1535.
16. R. M. Walmsley, D. J. C. Gardner and S. G. Oliver, Mol. Gen. Genet., 1983, 192, 361.
17. S.-H. Park and R. T. Raines, Protein Sci., 1997, 6, 2344.
18. A. W. Knight, N. J. Goddard, P. R. Fielden, M. G. Barker, N. Billinton and R. M. Walmsley, Meas. Sci. Technol., 1999, 10, 211.