Description: The SNAP-tag® and CLIP-tag™ are novel tools for the specific, covalent attachment of virtually any molecule to a protein of interest, providing simplicity and extraordinary versatility to the imaging of proteins in live and fixed cells, and to the study of proteins in vitro. The creation of a single gene construct yields a tagged fusion protein capable of forming a covalent linkage to a variety of functional groups, including fluorophores, biotin, or beads. This system provides a powerful and unique tool to study the role of proteins in a variety of highly dynamic processes, including protein trafficking, turnover and complex formation.
The CLIP-tag is a 20 kDa mutant of the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) that reacts specifically and rapidly with benzylcytosine (BC) derivatives, leading to covalent labeling of the CLIP-tag with a synthetic probe (Figure 1). The CLIP-tag has a number of features that make it ideal for a variety of protein labeling applications. The rate of the reaction of the CLIP-tag with these derivatives is largely independent of the nature of the synthetic probe attached to BC, permitting the labeling of CLIP fusion proteins with a wide variety of functional groups. Many of these CLIP-tag substrates are non-cell-permeable, allowing live-cell imaging of protein expression and localization on the cell surface (Figure 2). The ability to turn on the signal at will, together with the availability of a nonfluorescent blocking agent (CLIP-Cell™ Block), allows time-resolved pulse-chase analysis of protein trafficking to the cell surface, as well as subsequent internalization. Finally, the availability of orthogonal protein labeling systems from NEB permits simultaneous labeling of multiple proteins in a single cell (SNAP-tag, another hAGT variant that reacts exclusively with O6-benzylguanine substrates, and the ACP/MCP tags, small protein tags which can be enzymatically labeled on the cell surface with Coenzyme A derivatives).
The CLIP-Surface Starter Kit contains a mammalian expression plasmid (pCLIPf) encoding the CLIP-tag flanked by restriction sites for cloning a gene of interest, and two non-cell-permeable fluorescent CLIP-tag substrates. A positive control plasmid (pCLIPf-NK1R), encoding a CLIP-tagged protein (neurokinin-1 receptor) with a well-characterized cell surface localization, is also included. Lastly, a negative control “blocking agent” (CLIP-Cell™ Block) is included that interacts with the CLIP-tag, but is not fluorescent. There are two steps to using this system: subcloning and expression of the protein
of interest as a CLIPf fusion, and labeling of the fusion with the CLIP-tag substrate of choice.
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