Efficient proximity labeling in living cells and organisms with TurboID

Branon, Tess C; Bosch, Justin A.; Sanchez, Ariana D.; Udeshi, Namrata D.; Svinkina, Tanya; Carr, Steven A.; Feldman, Jessica L.; Perrimon, Norbert; Ting, Alice Y.

doi:10.1038/nbt.4201

Cited by 1,447 publications

(1,754 citation statements)

References 63 publications

Supporting

Mentioning

1,551

Contrasting

Unclassified

Order By: Relevance

“…Novel or improved applications using BioID ligase have spurred numerous follow-up articles including a smaller version of BioID with improved sensitivity and localization (22), its use for identifying protein-RNA interactions (23), split-BioID studies (24,25), and faster versions of BioID (26). Several conventional approaches have been utilized to stably introduce BioID-fusion proteins to cells including transfection (7), viral infection (27), and more recently, CRISPR-Cas9 (28).…”

Section: Introductionmentioning

confidence: 99%

BioID as a Tool for Protein-Proximity Labeling in Living Cells

Sears

May

Roux

2019

Methods in Molecular Biology

107

View full text Add to dashboard Cite

BioID has become an increasingly utilized tool for identifying candidate protein-protein interactions (PPIs) in living cells. This method utilizes a promiscuous biotin ligase, called BioID, fused to a protein-of-interest that when expressed in cells can be induced to biotinylate interacting and proximate proteins over a period of hours, thus generating a history of protein associations. These biotinylated proteins are subsequently purified and identified via mass spectrometry. Compared to other conventional methods typically used to screen strong PPIs, BioID allows for the detection of weak and transient interactions within a relevant biological setting over a defined period of time. Here we briefly review the scientific progress enabled by the BioID technology, detail an updated protocol for applying the method to proteins in living cells, and offer insights for troubleshooting commonly encountered setbacks.

show abstract

Section: Introductionmentioning

confidence: 99%

BioID as a Tool for Protein-Proximity Labeling in Living Cells

Sears

May

Roux

2019

Methods in Molecular Biology

107

View full text Add to dashboard Cite

show abstract

“…; Branon et al . ). The engineered ascorbate peroxidase APEX2 can be temporally controlled by the addition of H 2 O 2 , and catalyses the oxidation of exogenously supplied biotin–phenol (Lam et al .…”

Section: Inflammasomesmentioning

confidence: 97%

From atoms to physiology: what it takes to really understand inflammasomes

Schmidt

2019

The Journal of Physiology

View full text Add to dashboard Cite

Rapid inflammatory responses to cytosolic threats are mediated by inflammasomes – large macromolecular signalling complexes that control the activation of the pro‐inflammatory cytokines interleukin (IL)‐1β and IL‐18, as well as cell death by pyroptosis. Different inflammasome sensors are activated by diverse direct and indirect signals, and subsequently nucleate the polymerization of the adaptor molecule ASC to form signalling platforms macroscopically observed as ASC specks. Caspase‐1 is autocatalytically activated at these sites and subsequently matures pro‐inflammatory cytokines and the pore‐forming effector molecule gasdermin D. While most molecules and basic assembly principles have been deduced from reductionist experimental systems, we still lack fundamental information on the structure and regulation of these complexes in their physiological environment and in the interplay with other signalling pathways. In this review, novel experimental approaches are proposed, including some that rely on nanobodies and single domain antibodies, to understand inflammasome assembly and regulation in the context of the relevant tissues or cells.

show abstract

“…dCas9-BirA* (CasID) fusion extends this method with sequence-specific localization and biotin labeling by programmable single-guide RNA (sgRNA) (Schmidtmann, Anton, Rombaut, Herzog, & Leonhardt, 2016). While the radius of biotin ligation using BirA* is better defined (~20–30nm) than enrichment by cross-linking, the long reaction time usually necessary for this proximity-labeling method is unlikely to capture proteins only transiently associated to the target locus, though recent advances are ameliorating this limitation (Branon et al, 2018). …”

Section: Introductionmentioning

confidence: 99%

Adapting dCas9-APEX2 for subnuclear proteomic profiling

Gao

Rodríguez

Sontheimer

2019

Methods in Enzymology

View full text Add to dashboard Cite

Genome organization and subnuclear protein localization are essential for normal cellular function and have been implicated in the control of gene expression, DNA replication, and genomic stability. The coupling of chromatin conformation capture (3C), chromatin immunoprecipitation and sequencing, and related techniques have continuously improved our understanding of genome architecture. To profile site-specifically DNA-associated proteins in a high-throughput and unbiased manner, the RNA-programmable CRISPR–Cas9 platform has recently been combined with an enzymatic labeling system to allow proteomic landscapes at repetitive and nonrepetitive loci to be defined with unprecedented ease and resolution. In this chapter, we describe the dCas9-APEX2 experimental approach for specifically targeting a DNA sequence, enzymatically labeling local proteins with biotin, and quantitatively analyzing the labeled proteome. We also discuss the optimization and extension of this pipeline to facilitate its use in understanding nuclear and chromosome biology.

show abstract

Efficient proximity labeling in living cells and organisms with TurboID

Cited by 1,447 publications

References 63 publications

BioID as a Tool for Protein-Proximity Labeling in Living Cells

BioID as a Tool for Protein-Proximity Labeling in Living Cells

From atoms to physiology: what it takes to really understand inflammasomes

Adapting dCas9-APEX2 for subnuclear proteomic profiling

Contact Info

Product

Resources

About