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Journal Article

Snap-, CLIP- and Halo-tag labelling of budding yeast cells.

MPS-Authors
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Stagge,  F.
Department of NanoBiophotonics, MPI for biophysical chemistry, Max Planck Society;

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Mitronova,  G.
Department of NanoBiophotonics, MPI for biophysical chemistry, Max Planck Society;

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Belov,  V. N.
Department of NanoBiophotonics, MPI for biophysical chemistry, Max Planck Society;

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Wurm,  C. A.
Department of NanoBiophotonics, MPI for biophysical chemistry, Max Planck Society;

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Jakobs,  S.
Research Group of Mitochondrial Structure and Dynamics, MPI for biophysical chemistry, Max Planck Society;

Fulltext (public)

1857754.pdf
(Publisher version), 976KB

Supplementary Material (public)

1857754-Suppl-1.tif
(Supplementary material), 86KB

1857754-Suppl-2.tif
(Supplementary material), 3MB

1857754-Suppl-3.tif
(Supplementary material), 712KB

1857754-Suppl-4.tif
(Supplementary material), 833KB

1857754-Suppl-5.tif
(Supplementary material), 850KB

1857754-Suppl-6.tif
(Supplementary material), 3MB

1857754-Suppl-7.doc
(Supplementary material), 42KB

Citation

Stagge, F., Mitronova, G., Belov, V. N., Wurm, C. A., & Jakobs, S. (2013). Snap-, CLIP- and Halo-tag labelling of budding yeast cells. PLoS One, 8(10): e78745. doi:10.1371/journal.pone.0078745.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0014-B400-8
Abstract
Fluorescence microscopy of the localization and the spatial and temporal dynamics of specifically labelled proteins is an indispensable tool in cell biology. Besides fluorescent proteins as tags, tag-mediated labelling utilizing self-labelling proteins as the SNAP-, CLIP-, or the Halo-tag are widely used, flexible labelling systems relying on exogenously supplied fluorophores. Unfortunately, labelling of live budding yeast cells proved to be challenging with these approaches because of the limited accessibility of the cell interior to the dyes. In this study we developed a fast and reliable electroporation-based labelling protocol for living budding yeast cells expressing SNAP-, CLIP-, or Halo-tagged fusion proteins. For the Halo-tag, we demonstrate that it is crucial to use the 6'-carboxy isomers and not the 5'-carboxy isomers of important dyes to ensure cell viability. We report on a simple rule for the analysis of H-1 NMR spectra to discriminate between 6'- and 5'-carboxy isomers of fluorescein and rhodamine derivatives. We demonstrate the usability of the labelling protocol by imaging yeast cells with STED super-resolution microscopy and dual colour live cell microscopy. The large number of available fluorophores for these self-labelling proteins and the simplicity of the protocol described here expands the available toolbox for the model organism Saccharomyces cerevisiae.