ArticleThe Journal of biological chemistry2019
A robust and economical pulse-chase protocol to measure the turnover of HaloTag fusion proteins.
Article in The Journal of biological chemistry, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 30 papers.
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Who cites it
30 citing papers in PubMed.
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- Cancer degronopathies and protein-fate biomarkers in drug resistance and cancer progression: signalling mechanisms, measurement and translational limits.Frontiers in cell and developmental biology · 2026Review
- RNA-stabilized coat proteins for sensitive and simultaneous imaging of distinct single mRNAs in live cells.Nature methods · 2026Article
- Two-Color Timestamping of Gene Expression with a Chemigenetic Reporter System.Chembiochem : a European journal of chemical biology · 2025Article
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- Intracellular protein editing enables incorporation of noncanonical residues in endogenous proteins.Science (New York, N.Y.) · 2025Article
- Dual Labeling of Newly Synthesized Proteins and Existing Proteins UsingCurrent protocols · 2025Article
- Replication-dependent histone labeling dissects the physical properties of euchromatin/heterochromatin in living human cells.Science advances · 2025Article
- De novo missense variants in the PP2A regulatory subunit PPP2R2B in a neurodevelopmental syndrome: potential links to mitochondrial dynamics and spinocerebellar ataxias.Human molecular genetics · 2025Article
- Proteins required for stereocilia elongation during mammalian hair cell development ensure precise and steady heights during adult life.Proceedings of the National Academy of Sciences of the United States of America · 2024Article
- Inducing Receptor Degradation as a Novel Approach to Target CC Chemokine Receptor 2 (CCR2).International journal of molecular sciences · 2024Article
- A genetically-encoded fluorescence-based reporter to spatiotemporally investigate mannose-6-phosphate pathway.Molecular biology of the cell · 2024Article
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Authors and funding
6 authors.
Funding
Abstract
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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.