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Targeting and tracing antigens in live cells with fluorescent nanobodies


We fused the epitope-recognizing fragment of heavy-chain antibodies from Camelidae sp. with fluorescent proteins to generate fluorescent, antigen-binding nanobodies (chromobodies) that can be expressed in living cells. We demonstrate that chromobodies can recognize and trace antigens in different subcellular compartments throughout S phase and mitosis. Chromobodies should enable new functional studies, as potentially any antigenic structure can be targeted and traced in living cells in this fashion.

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Figure 1: Generation and characterization of a GFP-binding chromobody.
Figure 2: Recognition of subcellular structures by chromobodies raised against endogenous epitopes.
Figure 3: In vivo binding properties of various chromobodies analyzed by FRAP.

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We thank R.Y. Tsien, J. Ellenberg and K.F. Sullivan for providing expression vectors and cell lines, and J. Füner for the alpaca immunization. This work was supported by grants from the Deutsche Forschungsgemeinschaft to M.C.C. and H.L.

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Correspondence to Heinrich Leonhardt.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Schematic comparison of a conventional IgG and a heavy-chain IgG from Camelidae and various antigen-binding fragments derived thereof. (PDF 111 kb)

Supplementary Fig. 2

The GFP—chromobody is present as a stable monomeric protein in mammalian cells. (PDF 997 kb)

Supplementary Fig. 3

Schematic representation of the fusion proteins used in this study. (PDF 46 kb)

Supplementary Fig. 4

Antigen tracing with chromobodies. (PDF 363 kb)

Supplementary Methods (PDF 430 kb)

Supplementary Video 1

Tracing of a chromatin protein throughout mitosis. Time lapse imaging of a HeLa cell stably expressing histone H2B-GFP transfected with GFP—chromobody. At the time imaging was started (0 h), this cell was in late G2 phase. (MOV 1405 kb)

Supplementary Video 2

Tracing of a component of the replication machinery throughout S phase until G2. Time lapse imaging of cotransfected HeLa cells expressing GFP—PCNA and GFP—chromobody. At the start of imaging (0 h), the cells were in early to mid S phase. (MOV 1713 kb)

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Rothbauer, U., Zolghadr, K., Tillib, S. et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3, 887–889 (2006).

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