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Off the shelf proximity biotinylation using ProtA-TurboID

ABSTRACT: Proximity biotinylation is a commonly used method to identify the in vivo proximal proteome for proteins of interest. This technology typically relies on fusing a bait protein to a biotin ligase using overexpression or CRISPR-based tagging, thus prohibiting such assays in (primary) cell types that are difficult to transfect. We recently developed an ‘off the shelf’ proximity biotinylation method, which makes use of a recombinant enzyme consisting of the biotin ligase TurboID fused to the antibody-recognizing moiety Protein A. In this method, a bait specific antibody and the ProteinA-Turbo enzyme are consecutively added to permeabilized fixed or unfixed cells. Following incubation, during which ProteinA-Turbo-antibody-antigen complexes are formed, unbound molecules are washed away, after which bait-proximal biotinylation is triggered by the addition of exogenous biotin. Finally, biotinylated proteins are enriched from crude lysates using streptavidin beads followed by mass spectrometry-based protein identification. Here, we present a detailed protocol for this method

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Irene Santos Barriopedro

LAB HEAD: Prof Michiel Vermeulen

PROVIDER: PXD030963 | Pride | 2022-10-17

REPOSITORIES: Pride

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