Product Class: Kit

SNAP-Cell® Starter Kit
Item# Description List Price Web Price Qty
E9100S SNAP-Cell Starter Kit $413.00
$371.70
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*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

Product Introduction

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

Product Information

Description

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).

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 8A) 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 ReactionFigure 1: SNAP-tag Reaction

Figure 2:  Live cell imaging of SNAPf Fusion Proteins. 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.
Figure 3: Live cell imaging of SNAPf Fusion Proteins
Live COS-7 cells expressing mitochondrial cytochrome c 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
Product Categories:
Starter Kits Products,
Protein Labeling Products
Applications:
Snap Surface Products,
In vivo Imaging,
Pulse Chase
,
Receptor Internalization
,
Protein Localization
,
Protein Labeling Snap Clip

Properties & Usage

Materials Required but not Supplied

  • Mammalian Cell Lines 
  • DNA Transfection Reagents 
  • Standard Tissue Culture Media and Plasticware 
  • DMSO 
  • Hoechst 33342 for Nuclear Staining (optional)

Storage Temperature

-20°C

Product Notes

  1. 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.
  2. NEB 10-beta Competent E. coli (High Efficiency) (NEB #C3019) is recommended for propagating and subcloning of the vector and control plasmid.

Protocols, Manuals & Usage

Protocols

  1. Cellular Labeling (E9100)
  2. Use of SNAP-Cell Block with SNAP-Cell Substrates (E9100)
  3. Labeling of Proteins in vitro (E9100)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Application Notes

Tools & Resources

Selection Charts

  • Comparison of SNAP-tag®/CLIP-tag™ Technologies to GFP
  • Starter Kit Selection Chart

Faqs & Troubleshooting

FAQs

  1. What is the SNAP-tag®?
  2. How does it work?
  3. How specific is the binding of substrate to the SNAP-tag®?
  4. How does SNAP-tag labeling differ from using GFP fusion proteins?
  5. What linker type and length would you recommend?
  6. Can I clone my protein as a fusion to the N- or C-terminus of the tags?
  7. What is the smallest peptide and biggest protein you have cloned as SNAP-tag fusions?
  8. What is the solubility of SNAP-tag in insect and bacterial expression systems?
  9. What competent cell strains does NEB suggest for expression in E. coli?
  10. What competent cell E. coli strains are suitable for propagating SNAP-tag plasmids?
  11. Can SNAP-tag fusions be purified and refolded from inclusion bodies?
  12. Are substrates toxic to cells?
  13. How does SNAP-tag affect localization of the fusion partner?
  14. Can I use cell lines which express endogenous AGT?
  15. How stable is the labeled protein in mammalian cells?
  16. Are SNAP-tag substrates stable to fixation?
  17. Can cells expressing SNAP-tag be fixed prior to labeling?
  18. Can SNAP-tag be multiplexed with other protein labeling systems (GFP, Antibody)?
  19. Can you use SNAP-tag for in vivo FRET?
  20. Can cell-impermeable substrates be microinjected into cells, and how is the excess substrate exported?
  21. Does the SNAP-tag labeling reaction work in yeast?
  22. What happens to the fluorophore upon proteolysis?
  23. What conditions are recommended for SNAP-tag labeling in vitro?
  24. What conditions are incompatible with SNAP-tag labeling in vitro?
  25. Can SNAP-tag fusion proteins be labeled in a cell lysate?
  26. I have a compound that I would like to couple to a BG derivative. Where can I get advice?
  27. What is the difference between SNAP-tag and ACP-tag?
  28. What is the difference between SNAP- and CLIP-tag?
  29. Cellular Imaging and Analysis FAQs

Troubleshooting

  • Labeling with SNAP-tag® Technology Troubleshooting Guide

Quality, Safety & Legal

Quality Assurance Statement

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.

Specifications

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]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

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 [email protected].

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).