Pulse-chase experiment for the analysis of protein stability in cultured mammalian cells by covalent fluorescent labeling of fusion proteins.
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.
Yamaguchi
Kei
K
Laboratory of Human Gene Research, Department of Human Genome Research, Kazusa DNA Research Institute, Chiba, Japan.
Inoue
Shinichi
S
Ohara
Osamu
O
Nagase
Takahiro
T
D001483:Base Sequence; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D008967:Molecular Biology / Q000379:methods; D010957:Plasmids / Q000235:genetics; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins