SNAPtag

SNAP-tag is a self-labeling protein tag derived from the human O6-alkylguanine-DNA alkyltransferase (MGMT). It enables covalent attachment of a reporter or affinity probe to a protein of interest, by incubating cells or fixed samples with benzylguanine (BG) derivatives linked to fluorophores, biotin, or other labels. The tag is typically around 20 kDa and is encoded genetically so it can be fused to either terminus of a protein of interest for live-cell or fixed-sample labeling.

Mechanistically, SNAP-tag functions through the engineered MGMT active site, which catalyzes the transfer of the BG moiety from the BG substrate to a reactive cysteine within the tag. This covalent transfer forms a stable bond between the substrate and the SNAP-tag, resulting in irreversible labeling under physiological conditions. The reaction is highly specific for BG derivatives and can be performed in living cells, allowing temporal control of labeling through substrate addition.

Applications of SNAP-tag include live-cell imaging of protein localization and dynamics, pulse-chase experiments to track protein turnover, and multi-color labeling when used with different BG substrates. The system also supports purification strategies by using BG derivatives that carry affinity handles such as biotin, enabling selective isolation of SNAP-tagged proteins. Numerous BG substrates with a variety of fluorophores expand multiplex imaging possibilities, and labeling can be tuned by substrate concentration and exposure time.

Variants and related systems include SNAPf, a faster version with improved kinetics, and orthogonal tagging options such as CLIP-tag, which reacts with different substrate chemistries to enable simultaneous, separate labeling. SNAP-tag remains a widely used tool for covalent, modular labeling of proteins in cellular and molecular biology.