What is a SNAP-Tag?

The SNAP-tag is an enzyme based self-labeling protein tag. The SNAP-tag protein is a modified form of the human repair protein O6-alkylguanine-DNA-alkyltransferase (AGT), a 20 kDa protein. The SNAP-tag protein undergoes a self-labeling reaction to form a covalent bond with O6-benzylguanine derivatives. O6-Benzylguanine (BG) can be modified with a variety of reporter molecules such as fluorophores, peptides, or oligonucleotides. Using the SNAP-tag approach allows avoiding nonspecific labeling since most SNAP-tag substrates are chemically inert towards other proteins.

Figure 1: SNAP-tag protein bound to the substrate benzylguanine.

Figure 2: The reaction of SNAP-tag with O6-benzylguanine (BG) results in the covalent attachment of the label to the active site cysteine. If the SNAP-tag protein is fused or covalently attached to a protein of interest, a labeled BG substrate is used as a protein tag. Expression of proteins fused to the SNAP-tag in cells allows labeling of the selected proteins.

Linking the SNAP-tag to an oligonucleotide allows designing of hybridization probes. The SNAP-tag method can be used for DNA-mediated affinity assays such as immuno-PCR or proximity ligation assays (PLA) as well.

In 2013 Gu et al. reported the use of oligonucleotides in combination with the SNAP-tag approach for proximity-ligation-based applications.

In 2014, the SNAP-tag method was utilized for DNA-assisted protein analysis by Yan et al. 

Furthermore, self-labeling enzymes allow the design of molecular tools for confocal fluorescence microscopy (FM), super-resolution microscopy (SRM), and transmission electron microscopy (TEM). For these types of applications, a self-labeling protein such as the SNAP-tag, the Halo-Tag or the CLIP-Tag is reacted with a labeled ligand to form a covalent bond. Often the red fluorescent dye tetramethylrhodamine (TMR) is used as the fluorescent marker. However, other dyes or labels are used as well. These molecular tools used for correlative light and electron microscopy (CLEM) allow combining data on protein or protein complex dynamics with ultrastructural details in the low nanometer scale. However, for optimal use of these instruments, special molecular markers such as TMR conjugated probes are needed.

In 2014, Liss et al. showed that for CLEM, proteins of interest can be tagged with genetically encoded markers using self-labeling enzymes. Each tagging method employed a ligand conjugated to tetramethylrhodamine (TMR). Adding the TMR-ligand conjugates to living or fixed cells allowed covalently labeling of the tagged proteins of interest.

Design of releasable SNAP-tag probes

Incorporating a disulfide linker between the O6-benzylguanine and the rest of the substrate, for example, a fluorescent dye, allowed the design of releasable SNAP-tag probes. In 2012, Cole and Donaldson described the use of releasable SNP-tag probes for the study of endocytosis and recycling of proteins in cells.

Photoactivatable and photoconvertible fluorescence probes

Photoactivatable and photoconvertible fluorescence probes for protein labeling have also been utilized for selective coupling to SNAP-tag fusion proteins. As a prove-of-principle, Maurel et al. in 2010 reported that the photoactivatable Cy3 probe allows characterization of the mobility of a lipid-anchored cell surface protein and a G protein-coupled receptor (GPCR). This work illustrated the design principle for different photosensitive fluorophores with tailor-made properties for biomolecular imaging.


Cole NB, Donaldson JG. Releasable SNAP-tag Probes for Studying Endocytosis and Recycling. ACS Chemical Biology. 2012;7(3):464-469. doi:10.1021/cb2004252.

Cole NB. Site-Specific Protein Labeling with SNAP-Tags. Current protocols in protein science / editorial board, John E Coligan . [et al]. 2013;73:30.1.1-30.1.16. doi:10.1002/0471140864.ps3001s73.

Gautier A; Juillerat A; Heinis C; Corrêa IR Jr; Kindermann M; Beaufils F; Johnsson K (February 2008). "An engineered protein tag for multiprotein labeling in living cells". Chemistry and Biology. 15 (2): 128–136. PMID 18291317. doi:10.1016/j.chembiol.2008.01.007.

Gu GJ, Friedman M, Jost C, Johnsson K, Kamali-Moghaddam M, Plückthun A, Landegren U, Söderberg O.; Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation. N Biotechnol. 2013 Jan 25;30(2):144-52. doi: 10.1016/j.nbt.2012.05.005. Epub 2012 Jun 2.

Viktoria Liss, Britta Barlag, Monika Nietschke & Michael Hensel; Self-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron microscopy. Scientific Reports 5, Article number: 17740. https://www.nature.com/articles/srep17740.

Yan J, Gu GJ, Jost C, Hammond M, Plückthun A, Landegren U, et al. (2014) A Universal Approach to Prepare Reagents for DNA-Assisted Protein Analysis. PLoS ONE 9(9): e108061. https://doi.org/10.1371/journal.pone.0108061.