ArticleNature protocols2015
Labeling proteins on live mammalian cells using click chemistry.
Article in Nature protocols, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 88 papers.
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Who cites it
88 citing papers in PubMed.
- Bioorthogonal Chemistry in Neurobiology: From Imaging to Proteomics.Chembiochem : a European journal of chemical biology · 2026Review
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- Site-Specific Protein Bioconjugation Through Cellular Incorporation of Noncanonical Amino Acids.Angewandte Chemie (International ed. in English) · 2026Review
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- Development of anChemical & biomedical imaging · 2026Article
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- Conformational dynamics of the HIV-1 envelope glycoprotein from CRF01_AE is associated with susceptibility to antibody-dependent cellular cytotoxicity.Journal of virology · 2026Article
- Bioorthogonal click-chemistry labeling and visualization of dual noncanonical amino acid-incorporated HIV-1 Env on intact virions.Methods in enzymology · 2026Article
- The asymmetric opening of HIV-1 Env by a potent CD4 mimetic enables anti-coreceptor binding site antibodies to mediate ADCC.Nature communications · 2025Article
- Noncanonical amino acid incorporation enables minimally disruptive labeling of stress granule and TDP-43 proteinopathy.bioRxiv : the preprint server for biology · 2025Article
- Self-cleavage of the GAIN domain of adhesion G protein-coupled receptors requires multiple domain-extrinsic factors.Nature communications · 2025Article
- Double-Click Strategy Combining CuAAC and (Thia-) Diels-Alder Reactions; Application Toward Peptide Labeling.Chemistry (Weinheim an der Bergstrasse, Germany) · 2025Article
- Quantitative Protein Labeling in Live Cells by Controlling the Redox State of Encoded Tetrazines.Journal of the American Chemical Society · 2025Article
- Click biology highlights the opportunities from reliable biological reactions.Nature chemical biology · 2025Review
- Identification of Actionable Targeted Protein Degradation Effector Sites through Site-Specific Ligand Incorporation-Induced Proximity (SLIP).Journal of the American Chemical Society · 2025Article
- Fluorescent labeling of proteins in vitro and in vivo using encoded peptide tags.The Journal of biological chemistry · 2025Review
- Comparative Study of Click Handle Stability in Common Ligation Conditions.Bioconjugate chemistry · 2025Article
- Neural stem and progenitor cells support and protect adult hippocampal function via vascular endothelial growth factor secretion.Molecular psychiatry · 2025Article
- A Single Bioorthogonal Reaction for Multiplex Cell Surface Protein Labeling.Journal of the American Chemical Society · 2025Article
28 more citing papers are in PubMed but not listed here.
Corrections and comments
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Authors and funding
5 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.
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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.