Protocol

Reverse Chemical Genetics

Volume 577 of the series Methods in Molecular Biology™ pp 121-131

Date:

Pulse-Chase Experiment for the Analysis of Protein Stability in Cultured Mammalian Cells by Covalent Fluorescent Labeling of Fusion Proteins

  • Kei YamaguchiAffiliated withLaboratory of Human Gene Research, Department of Human Genome Research, Kazusa DNA Research Institute
  • , Shinichi InoueAffiliated withLaboratory of Human Gene Research, Department of Human Genome Research, Kazusa DNA Research Institute
  • , Osamu OharaAffiliated withDepartment of Human Genome Research, Kazusa DNA Research InstituteLaboratory for Immunogenomics, RIKEN Research Institute for Allergy and Immunology
  • , Takahiro NagaseAffiliated withLaboratory of Human Gene Research, Department of Human Genome Research, Kazusa DNA Research Institute

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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.

Key words

Covalent labeling E3 ubiquitin ligase Fluorescence Imaging Intracellular Protein degradation signal Protein stability Pulse chase