DescriptionThe 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.
- pSNAPf Vector
- pSNAPf-Cox8A Control Plasmid
- SNAP-Cell® 505
- SNAP-Cell® TMR-Star
- SNAP-Cell® Block
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|
Properties and Usage
Materials Required but not Supplied
- Mammalian Cell Lines
- DNA Transfection Reagents
- Standard Tissue Culture Media and Plasticware
- Hoechst 33342 for Nuclear Staining (optional)
Notice To Buyer/User: The Buyer/User has a non-exclusive license to use this system or any component thereof for RESEARCH AND DEVELOPMENT PURPOSES ONLY. Commercial use of this system or any components thereof requires a license from New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938.
Cellular Imaging and Analysis (i.e., SNAP and CLIP products)
Notice to Buyer/User: The Buyer/User has a non-exclusive license to use this system or any component thereof for RESEARCH AND DEVELOPMENT ONLY. Commercial use of this system or any components thereof requires a license from New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938.
The products and/or their use may be covered by one or more of the following patents and patent applications:
Methods for Using O6-Alkylguanine-DNA-Alkyltransferases: US 7,939,284; US 8,367,361; EP 1 410 023; EP 2 037 271; EP 1 696 234; EP 2 211 177; JP 4195815; JP 4226053; CN 1295510; CN 1975422; and SG 100125.
Substrates for O6-Alkylguanine-DNA Alkyltransferase: US 7,799,524; and JP 4976651.
Mutants of O6-Alkylguanine-DNA-Alkyltransferases: US 7,888,090. EP 1720981 (pending).
Specific Substrates for O6-Alkylguanine-DNA-Alkyltransferases: US 8,163,479. EP 1730298 (pending).
- 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.
- What is the SNAP-tag®?
- How does it work?
- How specific is the binding of substrate to the SNAP-tag®?
- How does SNAP-tag labeling differ from using GFP fusion proteins?
- What linker type and length would you recommend?
- Can I clone my protein as a fusion to the N- or C-terminus of the tags?
- What is the smallest peptide and biggest protein you have cloned as SNAP-tag fusions?
- What is the solubility of SNAP-tag in insect and bacterial expression systems?
- What competent cell strains does NEB suggest for expression in E. coli?
- What competent cell E. coli strains are suitable for propagating SNAP-tag plasmids?
- Can SNAP-tag fusions be purified and refolded from inclusion bodies?
- Are substrates toxic to cells?
- How does SNAP-tag affect localization of the fusion partner?
- Can I use cell lines which express endogenous AGT?
- How stable is the labeled protein in mammalian cells?
- Are SNAP-tag substrates stable to fixation?
- Can cells expressing SNAP-tag be fixed prior to labeling?
- Can SNAP-tag be multiplexed with other protein labeling systems (GFP, Antibody)?
- Can you use SNAP-tag for in vivo FRET?
- Can cell-impermeable substrates be microinjected into cells, and how is the excess substrate exported?
- Does the SNAP-tag labeling reaction work in yeast?
- What happens to the fluorophore upon proteolysis?
- What conditions are recommended for SNAP-tag labeling in vitro?
- What conditions are incompatible with SNAP-tag labeling in vitro?
- Can SNAP-tag fusion proteins be labeled in a cell lysate?
- I have a compound that I would like to couple to a BG derivative. Where can I get advice?
- What is the difference between SNAP-tag and ACP-tag?
- What is the difference between SNAP- and CLIP-tag?
- Cellular Imaging and Analysis FAQs