Rapid FlAsH labelling in the budding yeast Saccharomyces cerevisiae.
Live cell imaging of protein distributions is an essential tool in modern cell biology. It relies on the functional labelling of a host protein with a fluorophore, which may either be a genetically fused fluorescent protein or an organic dye binding to the host protein. The biarsenical-tetracysteine system or 'FlAsH-labelling', is based on the high affinity interaction between a biarsenical probe and a small protein tag. This approach has been successfully used for live cell imaging in the budding yeast Saccharomyces cerevisiae. However, the established labelling protocols require a lengthy overnight incubation of the cells with the dye under tightly controlled growth conditions, which severely limits the use of this approach. In this study, we characterize an efficient method for introducing FlAsH-EDT(2) into live budding yeast cells using standard electroporation. The labelling time is reduced from more than 12 h to less than 1 h without compromising the labelling efficiency or cell viability. This approach may be used for cells in different growth phases or grown under different conditions. It may be further extended to other small high affinity probes, thus opening up new possibilities for labelling in budding yeast.
Mitochondrial Structure and Dynamics Group, Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
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Wurm
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Suppanz
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Stoldt
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Jakobs
D000595:Amino Acid Sequence; D003545:Cysteine / Q000032:analysis / Q000737:chemistry; D018274:Electroporation; D005456:Fluorescent Dyes; D008928:Mitochondria / Q000648:ultrastructure*; D010455:Peptides / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000166:cytology*; D013194:Staining and Labeling / Q000379:methods*
D005456:Fluorescent Dyes; D010455:Peptides; D003545:Cysteine