Abstract
The ability to specifically label proteins with a wide range of optical properties and functionalities can help reveal information about protein functions and dynamics in living cells. Here, we describe a technology for covalent tethering of organic probes directly to a specially designed reporting protein expressed in live cells. The reporting protein can be used in a manner similar to green fluorescent protein, except that the fluorophore might be interchanged among a variety of standard dyes. This allows living cells to be imaged at different wavelengths without requiring changes to the underlying genetic constructs, and the colors can be rapidly switched to allow temporal analysis of protein fate. The stability of the bond permits imaging of live cells during long time periods, imaging of fixed cells, and multiplexing with different cell/protein analysis techniques. The dyes can also be exchanged with other functional molecules, such as biotin to serve as an affinity handle, or even solid supports for direct covalent immobilization. The technology complements existing methods and provides new options for cell imaging and protein analysis.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Los, G. et al. (2005) HaloTag™ interchangeable labeling technology for cell imaging and protein capture. Cell Notes 11, 2–6.
Kalderon, D., Roberts, B. L., Richardson, W. D., and Smith, A. E. (1984) A short amino acid sequence able to specify nuclear location. Cell 39(3 Part 2), 499–509.
Ghosh, S., May, M. J., and Kopp, E. B. (1998) NF-κB and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16, 225–260.
Karin, M., Yamamoto, Y., and Wang, Q. M. (2004) The IKK NF-kappa B system: a treasure trove for drug development. Nature Rev. 3, 17–26.
Burnstein, E. and Duckett, C. S. (2003) Dying for NF-kappaB? Control of cell death by transcriptional regulation of the apoptotic machinery. Curr. Opin. Cell Biol. 15, 732–737.
Schmid, J. A., Birbach, A., Hofer-Warbinek, R., et al. (2000) Dynamics of NF kappa B and Ikappa Balpha studied with green fluorescent protein (GFP) fusion proteins. Investigation of GFP-p65 binding to DNa by fluorescence resonance energy transfer. J Biol. Chem. 275(22), 17,035–17,042.
Freshney, R. I. (1986) Animal Cell Culture: A Practical Approach, IRL Press, Oxford, UK.
Ausubel, F. M. et al. (1994) Current Protocols in Molecular Biology, Green Publishing Associates and Wiley-Interscience, New York.
Felgner, P. L., Holm, M., and Chan, H. (1989) Cationic liposome mediated transfection. Proc. West. Pharmacol. Soc. 32, 115–216.
Felgner, P. L. and Ringold, G. M. (1989) Cationic liposome-mediated transfection. Nature 337, 387–388.
Chen, C. and Okayama, H. (1987) High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell Biol. 7, 2745–2752.
Wigler, M. et al. (1977) Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell 11, 223–232.
Chu, G., Hayakawa H., and Berg, P. (1987) Electroporation for the efficient transfection of mammalian cells with DNA. Nucl. Acids Res. 15, 131–226.
Shikegawa, K. and Dower, W. J. (1988) Electroporation of eukaryotes and prokaryotes: a general approach to the introduction of macromolecules into cells. Biotechniques 6, 742–751.
Ramezani, A. and Hawley, R. G. (2002) Overview of the HIV-1 lentiviral vector system, in Current Protocols in Molecular Biology, (Ausubel, F.M. et al., ed.), John Wiley and Sons, Inc., Hoboken, NJ, pp. 16.21.1–16.21.15.
Moss, B. and Earl, P. (1998) Overview of the vaccinia virus expression system, in Current Protocols in Molecular Biology, (Ausubel, F.M. et al., ed.) John Wiley and Sons, Inc., Hoboken, NJ, pp. 16.15.1–16.15.5.
Herman, B. (2001) Fluorescence Microscopy, 2nd ed., Springer-Verlag, New York.
Perassamy, A., ed. (2001) Methods in Cellular Imaging, Oxford University Press, New York.
Kulakova, A. N., Larkin, M. J., and Kulakov, L. A. (1997) The plasmid-located haloalkane dehalogenase gene from Rhodococcus rhodochrous NCIMB 13064. Microbiology 143, 109–115.
Schindler, J. F. et al. (1999) Haloalkane dehalogenase: steady-state kinetics and halide inhibition. Biochemistry 38, 5772–5778.
Newman, J. et al. (1999) Haloalkane dehalogenase: structure of a Rhodococcus enzyme. Biochemistry 38, 16,105–16,114.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press, Inc.
About this protocol
Cite this protocol
Los, G.V., Wood, K. (2007). The HaloTag™. In: Taylor, D.L., Haskins, J.R., Giuliano, K.A. (eds) High Content Screening. Methods in Molecular Biology, vol 356. Humana Press. https://doi.org/10.1385/1-59745-217-3:195
Download citation
DOI: https://doi.org/10.1385/1-59745-217-3:195
Publisher Name: Humana Press
Print ISBN: 978-1-58829-731-0
Online ISBN: 978-1-59745-217-5
eBook Packages: Springer Protocols