The self-labeling protein HaloTag has been used extensively to determine the localization and turnover of proteins of interest at the single-cell level. To this end, halogen-substituted alkanes attached to diverse fluorophores are commercially available that allow specific, irreversible labeling of HaloTag fusion proteins; however, measurement of protein of interest half-life by pulse-chase of HaloTag ligands is not widely employed because residual unbound ligand continues to label newly synthesized HaloTag fusions even after extensive washing. Excess unlabeled HaloTag ligand can be used as a blocker of undesired labeling, but this is not economical. In this study, we screened several inexpensive, low-molecular-weight haloalkanes as blocking agents in pulse-chase labeling experiments with the cell-permeable tetramethylrhodamine HaloTag ligand. We identified 7-bromoheptanol as a high-affinity, low-toxicity HaloTag-blocking agent that permits protein turnover measurements at both the cell population (by blotting) and single-cell (by imaging) levels. We show that the HaloTag pulse-chase approach is a nontoxic alternative to inhibition of protein synthesis with cycloheximide and extend protein turnover assays to long-lived proteins.
Keywords: E3 ubiquitin ligase; HaloTag; fusion protein; haloalkane; haloalkane hydrolase; protein degradation; protein stability; protein turnover; pulse-chase experiment; self-labeling proteins; ubiquitin ligase.
© 2019 Merrill et al.