Efficient and Site-Specific Protein Bioconjugation Quantification in Mammalian Cells.

Gabriella Belsito, Olivia Shade, Alexander Deiters

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Article in Methods in molecular biology (Clifton, N.J.), 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.

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5 · Who and what money

Authors and funding

3 authors.

Gabriella BelsitoDepartment of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.

Olivia ShadeDepartment of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.

Alexander DeitersDepartment of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA. deiters@pitt.edu.

Funding

No grant is acknowledged in the PubMed record.

6 · The paper itself

Abstract

Techniques for the quantification of protein bioconjugation in live cells have been limited. This new method allows for quantification to be measured in cellulo. Inverse electron-demand Diels-Alder (IEDDA) cycloaddition reactions are utilized to label biomolecules. IEDDA compatible unnatural amino acids (UAAs) and their chloroalkane-derivatized conjugates are labeled with the HaloTag enzyme, which is used to quantify the bioconjugation reaction through a gel shift of the conjugated protein of interest. This method is efficient and able to be adjusted to the specific protein of interest. It utilizes common instrumentation and techniques that are found in most labs. This tool's versatility is highlighted by the fact that a variety of UAA/bioconjugation pairs can be used at different incorporation sites in any protein of interest.

Indexed as

ProteinsAmino AcidsAnimalsClick ChemistryCycloaddition ReactionHumansStaining and LabelingAmino AcidsProteinsClick chemistryGenetic code expansionProtein bioconjugationTetrazine ligation

Identifiers

PMID42542482

DOI10.1021/jacs.5b06847

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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.