The SNAP-tag® is a novel tool 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 SNAP-tag is a 20 kDa mutant of the human DNA repair protein O6-alkylguanine- DNA alkyltransferase (hAGT) that reacts specifically and rapidly with benzylguanine (BG) and benzylchloropyrimidine (CP) derivatives, leading to covalent labeling of the SNAP-tag with a synthetic probe (Figure 1). The SNAP-tag has a number of features that make it ideal for a variety of protein labeling applications. The rate of the reaction of the SNAP-tag with these derivatives is largely independent of the nature of the synthetic probe attached to BG, permitting the labeling of SNAP fusion proteins with a wide variety of functional groups. Many of these SNAP-tag substrates are cell-permeable, allowing live-cell imaging of protein expression and localization (Figure 2). The ability to turn on the signal at will, together with the availability of a cell-permeable nonfluorescent blocking agent (SNAP-Cell® Block), allows time-resolved pulse-chase analysis of protein trafficking. Finally, the availability of orthogonal protein labeling systems from NEB permits simultaneous labeling of multiple proteins in a single cell (CLIP-tag™, a SNAP-tag variant that reacts exclusively with O2-benzylcytosine substrates, and the ACP/MCP tags, small protein tags which can be enzymatically labeled on the cell surface with Coenzyme A derivatives).
The SNAP-Cell Starter Kit contains a mammalian expression plasmid (pSNAPf) encoding the SNAP-tag flanked by restriction sites for cloning a gene of interest, and two cell-permeable fluorescent SNAP-tag substrates. A positive control plasmid (pSNAPf-Cox8A), encoding a SNAP-tagged protein (cytochrome c oxidase) with a well-characterized mitochondrial localization, is also included. Lastly, a negative control “blocking agent” (SNAP-Cell Block) is included that interacts with the SNAP-tag, but is not fluorescent. There are two steps to using this system: subcloning and expression of the protein of interest as a SNAPf fusion, and labeling of the fusion with the SNAP-tag substrate of choice.
Figure 1:SNAP-tag Reaction Figure 2: Live cell imaging of SNAPf Fusion Proteins. Live CHO-K1 cells transiently transfected with pSNAPf-H2B. Cells were labeled with SNAP-Cell 505-star (green) for 15 minutes at 37°C, 5% CO2.Figure 3: Live cell imaging of SNAPf Fusion Proteins. Live COS-7 cells expressing mitochondrial cytochrome oxidase 8A-SNAPf (SNAPf-Cox8A) were labeled with SNAP-Cell TMR-Star (red). Nuclei were counterstained with Hoechst 33342 (blue).
Kit Components
The following reagents are supplied with this product:
Store at (°C)
Concentration
pSNAPf Vector
-20
0.5 mg/ml
pSNAPf-Cox8A Control Plasmid
-20
0.5 mg/ml
SNAP-Cell® 505-Star
10 nmol
SNAP-Cell® TMR-Star
-20
6 nmol
SNAP-Cell® Block
-20
20 nmol
Notes:
For long-term storage, all kit components should be stored at -20˚C. Plasmid solutions can be stored at 4˚C for up to one week. Undissolved dye and blocking substrates can be stored at 4˚C for up to 4 weeks protected from light and moisture. With proper storage at -20°C the substrates should be stable for at least three years dry or 3 months dissolved in DMSO.NEB 10-beta Competent E. coli (High Efficiency) (NEB #C3019) is recommended for propagating and subcloning of the vector and control plasmid.
