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.