Abstract
A system to use fluorometry for the search of protein crystallization buffers was developed. The screening of candidates was done with a use of short gel-filtration column (Sephacryl S-100 HR) linked to the fluorometer. Protein elution was monitored by intrinsic fluorescence or emission from its labels. This method was applied to actin and actin complexes. When nuclei were formed in actin solution, preceding the peak of actin, a new peak appeared on the elution curve. It was found that the fluorescence allows detection of even small amount of nuclei formed in the buffer. Using the screened buffers, crystal growths were attempted. The images of crystals were captured by fluorescence microscope. The monitoring of nuclei with this method will accelerate the screening of crystallization buffers. This system is applicable to the crystallization of other proteins and their complexes.
Similar content being viewed by others
REFERENCES
Z. Kam, H. B. Shore, and G. Feher (1978). J. Mol. Biol. 123, 539–555.
M. Skouri et al. (1991) FEBS Lett. 295, 84–88.
A. R. Ferre-D'Amare and S. K. Burley (1997) Meth. Enzymolo. 276, 157–166.
J. D. Donovan, G. B. Benedek, and M. C. Carey (1987) Biochemistry 26, 8116–8125.
W. Kadima et al. (1990) Biophys. J. 57, 125–132.
J. A. Cooper and T. D. Pollard Methods Enzymol. 85 182–233.
T. Ikkai and K. Kondo (2000) IUBMB Life 49, 77–79.
T. Ikkai and H. Kondo (1995) Biochem. Mol. Biol. Int. 37, 1153–1161.
K. Kashima, M. Seidou, and Y Kito (1978) Biochim. Biophys. Acta 536, 78–87.
T. Ikkai and H. Kondo (1996) J. Biochem. Biophys. Meth. 33,55–58.
L. D. Selden, H. J. Kinosian, J. E. Estes and L. C. Gershman (1994) in J. E. Estes and P. J. Higgins (Eds.) Advances in Experimental Medicine and Biology, pp. 51–57 Plenum Press, New York.
T. Ikkai and K. Shimada (2000) J. Fluoresc 10, 77–79.
W. Kabsch, H. G. Mannherz, D. Suck, E. F. Pai, and K. C. Holmes (1990) Nature 347, 37–44.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ikkai, T., Shimada, K. Introduction of Fluorometry to the Screening of Protein Crystallization Buffers. Journal of Fluorescence 12, 167–171 (2002). https://doi.org/10.1023/A:1016892213213
Issue Date:
DOI: https://doi.org/10.1023/A:1016892213213