UNIT 30.1 Site-Specific Protein Labeling with SNAP-Tags

ABSTRACT

Site-specific labeling of cellular proteins with chemical probes is a powerful tool for studying protein function in living cells. A number of small peptide and protein tags have been developed that can be labeled with synthetic probes with high efficiencies and specificities and provide flexibility not available with fluorescent proteins. The SNAP-tag is a modified form of the DNA repair enzyme human O⁶-alkylguanine-DNA-alkyltransferase, and undergoes a self-labeling reaction to form a covalent bond with O⁶-benzylguanine (BG) derivatives. BG can be modified with a wide variety of fluorophores and other reporter compounds, generally without affecting the reaction with the SNAP-tag. In this unit, basic strategies for labeling SNAP-tag fusion proteins, both for live cell imaging and for in vitro analysis, are described. This includes a description of a releasable SNAP-tag probe that allows the user to chemically cleave the fluorophore from the labeled SNAP-tag fusion. In vitro labeling of purified SNAP-tag fusions is briefly described. Curr. Protoc. Protein Sci. 73:30.1.1-30.1.16. © 2013 by John Wiley & Sons, Inc.