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Multicolor in vitro translation

Nature Biotechnology volume 21, pages 1093–1097 (2003)Cite this article

Abstract

In vitro translation is a widely used tool for both analytical and preparative purposes. For analytical purposes, small amounts of proteins are synthesized and visualized by detection of labeled amino acids incorporated during translation. The original strategy of incorporating radioactively labeled amino acids, such as [35S]methionine or [14C]leucine, has been superseded by the addition of antigenic tags or the incorporation of biotin-labeled or BODIPY-FL–labeled amino acids. Such nonradioactive tags are easier to visualize after translation and do not pose a radiation hazard. Among the nonradioactive tags, BODIPY-FL–lysine offers the advantage that proteins that have incorporated this amino acid can be directly visualized after gel electrophoresis. We show here that multiple fluorophores introduced into proteins can considerably extend their usefulness, particularly for the comparison of in vitro–translated proteins from related sources. This technology can be applied in various situations, including the simplified detection of rare truncating mutations in clinical samples from cancer patients.

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Figure 1: Multicolor digital protein truncation.
Figure 2: Unique spectral properties of the four fluorophores.
Figure 3: Examples of the multicolor digital protein truncation test conducted on fecal DNA samples.

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Acknowledgements

We thank Dan Simpson and Dipayan Chaudhuri for expert technical assistance. G.T. is a recipient of a Junior Research Fellowship from Trinity College, Cambridge, UK. This work was supported by the National Colorectal Cancer Research Alliance, by the Caroline Law Fund, by the University of Texas M.D. Anderson Cancer Center, by the Clayton Fund, and by grants (CA 62924, CA 43460, CA 573345 and GM 07184) from the US National Institutes of Health.

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Author notes

  1. Giovanni Traverso and Frank Diehl: These authors contributed equally to this work.

Authors and Affiliations

  1. Howard Hughes Medical Institute, Sidney Kimmel Comprehensive Cancer Center, and Graduate Program in Human Genetics, Johns Hopkins School of Medicine, 1650 Orleans Street, Baltimore, 21231, Maryland, USA

    Giovanni Traverso, Frank Diehl, Kenneth W Kinzler & Bert Vogelstein

  2. Trinity College, Cambridge, CB21TQ, UK

    Giovanni Traverso

  3. Promega Corporation, 2800 Woods Hollow Road, Madison, 53711-5399, Wisconsin, USA

    Robin Hurst

  4. Exact Sciences, 63 Great Road, Maynard, 01754, Massachusetts, USA

    Anthony Shuber & Duncan Whitney

  5. Division of Cancer Prevention, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, 77030, Texas, USA

    Constance Johnson & Bernard Levin

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  1. Giovanni Traverso
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  8. Kenneth W Kinzler
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Corresponding author

Correspondence to Bert Vogelstein.

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Competing interests

Under agreements between the Johns Hopkins University and Exact Sciences, Inc., Genzyme Molecular Oncology, Inc. (Genzyme) and Hoffmann-LaRoche, Inc., G.T., K.W.K. and B.V. are entitled to a share of the royalties received by the university on sales of products related to the use of in vitro translation or stool DNA for cancer diagnosis. K.W.K. receives research funding from Genzyme and is a consultant to Exact Sciences and Genzyme. The university, K.W.K. and B.V. also own stock in Genzyme, which is subject to certain restrictions under university policy. The terms of these arrangements are being managed by the university in accordance with its conflict-of-interest policies. B.L. serves on the Scientific Advisory Board of Enterix and receives research support from Pfizer.

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Traverso, G., Diehl, F., Hurst, R. et al. Multicolor in vitro translation. Nat Biotechnol 21, 1093–1097 (2003). https://doi.org/10.1038/nbt857

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