A robust and economical pulse-chase protocol to measure the turnover of HaloTag fusion proteins

Ronald A Merrill; Jianing Song; Rikki A Kephart; Annette J Klomp; Claire E Noack; Stefan Strack

doi:10.1074/jbc.RA119.010596

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A robust and economical pulse-chase protocol to measure the turnover of HaloTag fusion proteins

Journal article

Open access

Peer reviewed

A robust and economical pulse-chase protocol to measure the turnover of HaloTag fusion proteins

Ronald A Merrill, Jianing Song, Rikki A Kephart, Annette J Klomp, Claire E Noack and Stefan Strack

The Journal of biological chemistry, Vol.294(44), pp.16164-16171

11/01/2019

DOI: 10.1074/jbc.RA119.010596

PMCID: PMC6827285

PMID: 31511325

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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.

Heptanol - analogs & derivatives

Staining and Labeling - methods

Fluorescent Dyes - metabolism

Half-Life

Biological Assay - methods

Rhodamines - metabolism

Proteins

Proteolysis

Single-Cell Analysis - methods

Ligands

Protein Stability

Rhodamines - chemistry

Heptanol - chemistry

Details

Title: Subtitle: A robust and economical pulse-chase protocol to measure the turnover of HaloTag fusion proteins
Creators: Ronald A Merrill - Department of Neuroscience and Pharmacology, and Iowa Neuroscience Institute, University of Iowa, Iowa City, Iowa 52242
Jianing Song - Department of Neuroscience and Pharmacology, and Iowa Neuroscience Institute, University of Iowa, Iowa City, Iowa 52242
Rikki A Kephart - Department of Neuroscience and Pharmacology, and Iowa Neuroscience Institute, University of Iowa, Iowa City, Iowa 52242
Annette J Klomp - Department of Neuroscience and Pharmacology, and Iowa Neuroscience Institute, University of Iowa, Iowa City, Iowa 52242
Claire E Noack - Department of Neuroscience and Pharmacology, and Iowa Neuroscience Institute, University of Iowa, Iowa City, Iowa 52242
Stefan Strack - Department of Neuroscience and Pharmacology, and Iowa Neuroscience Institute, University of Iowa, Iowa City, Iowa 52242 stefan-strack@uiowa.edu
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.294(44), pp.16164-16171
DOI: 10.1074/jbc.RA119.010596
PMID: 31511325
PMCID: PMC6827285
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: R21 NS087908 / NINDS NIH HHS R01 NS056244 / NINDS NIH HHS
Language: English
Date published: 11/01/2019
Academic Unit: Pathology; Iowa Neuroscience Institute; Neuroscience and Pharmacology
Record Identifier: 9984068362902771

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