Summary
We used HaloTag® labeling technology for the pulse labeling of proteins in cultured mammalian cells. HaloTag® technology allows a HaloTag-fusion protein to covalently bind to a specific small molecule fluorescent ligand. Thus specifically labeled HaloTag-fusion proteins can be chased in cells and observed in vitro after separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The Fluorescent HaloTag® ligand allows quantification of the labeled proteins by fluorescent image analysis. Herein, we demonstrated that the method allows analysis of the intracellular protein stability as regulated by protein-degradation signals or an exogenously expressed E3 ubiquitin ligase.
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Acknowledgments
This project was supported by a grant from the Chiba prefectural government. We are grateful to B. Bulleit for his valuable comments. We thank K. Ozawa, T. Watanabe, and K. Yamada for their technical assistance.
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Yamaguchi, K., Inoue, S., Ohara, O., Nagase, T. (2009). Pulse-Chase Experiment for the Analysis of Protein Stability in Cultured Mammalian Cells by Covalent Fluorescent Labeling of Fusion Proteins. In: Koga, H. (eds) Reverse Chemical Genetics. Methods in Molecular Biology™, vol 577. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-232-2_10
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DOI: https://doi.org/10.1007/978-1-60761-232-2_10
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