biostudies-literatureKeeble AHBiotechnology and Biological Sciences Research Council7281-7291https://www.ebi.ac.uk/biostudies/studies/S-EPMC7844731biostudies-literatureUnknown11(28)Proteins span an extraordinary range of shapes, sizes and functionalities. Therefore generic approaches are needed to overcome this diversity and stream-line protein analysis or application. Here we review SpyTag technology, now used in hundreds of publications or patents, and its potential for detecting and controlling protein behaviour. SpyTag forms a spontaneous and irreversible isopeptide bond upon binding its protein partner SpyCatcher, where both parts are genetically-encoded. New variants of this pair allow reaction at a rate approaching the diffusion limit, while reversible versions allow purification of SpyTagged proteins or tuned dynamic interaction inside cells. Anchoring of SpyTag-linked proteins has been established to diverse nanoparticles or surfaces, including gold, graphenChemical sciencePower to the protein: enhancing and combining activities using the Spy toolbox.PMC7844731BB/S007369/1Keeble AHHowarth MfalsePower to the protein: enhancing and combining activities using the Spy toolbox.Proteins span an extraordinary range of shapes, sizes and functionalities. Therefore generic approaches are needed to overcome this diversity and stream-line protein analysis or application. Here we review SpyTag technology, now used in hundreds of publications or patents, and its potential for detecting and controlling protein behaviour. SpyTag forms a spontaneous and irreversible isopeptide bond upon binding its protein partner SpyCatcher, where both parts are genetically-encoded. New variants of this pair allow reaction at a rate approaching the diffusion limit, while reversible versions allow purification of SpyTagged proteins or tuned dynamic interaction inside cells. Anchoring of SpyTag-linked proteins has been established to diverse nanoparticles or surfaces, including gold, graphen2020-01-01T00:00:00Z2020 Jul2025-04-05T12:02:39.742Z2021-02-21T08:32:22ZS-EPMC78447313355245910.1039/d0sc01878c