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 in addition to controls.
The kit provides all the components necessary for the expression and labeling of the gene of interest in mammalian cells
It contains a positive and a negative control
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 substrates provided are cell-permeable
The SNAP-tag is a 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:
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.
The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].)
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at gbd@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
Licenses
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).
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manualE9100