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  • CLIP-Surface™ Starter Kit

    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.


    Figure 1:
    Clip-tag Reaction.

    Figure 2:
    Live CHO-K1 cells transiently transfected with pCLIPf-NK1R. Cells were labeled with CLIP-Surface™ 488 (green) for 30 minutes and counterstained with Hoechst 33342 (blue).

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    pCLIPf Vector-200.5 mg/ml
    pCLIPf-NK1R Control Plasmid-200.5 mg/ml
    CLIP-Cell™ Block-2020 nmol
    CLIP-Surface™ 488-2010 nmol
    CLIP-Surface™ 547-2010 nmol

    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 two 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.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Manuals

    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].
    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
    1. Cellular Labeling (E9230)
    2. Use of CLIP-Cell Block with CLIP-Cell Substrates (E9230)
    3. Labeling of Proteins in Solution (E9230)

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    Application Notes