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  • CLIP-Cell™ Block

    Description

    CLIP-Cell™ Block (bromothenylcytosine, BTC) is a non-fluorescent compound that blocks the reactivity of the CLIP-tag™ in vitro and inside or on the surface of living cells. It can be used to generate inactive controls in live cell labeling experiments performed with CLIP-tag fusion proteins. BTC reacts with CLIP-tag irreversibly, inactivating it for subsequent labeling steps. 

    The CLIP-tag protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest. CLIP-tag is a protein tag based on human O6-alkylguanine-DNAalkyltransferase (hAGT). CLIP-tag substrates are derivatives of benzylcytosine (BC). In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the reactive cysteine of CLIP-tag forming a stable thioether bond.  Although CLIP-tag is based on the same protein as SNAP-tag®, the benzylcytosine substrates form a separate class of substrates, different from the benzylguanine substrates recognized by SNAP-tag. CLIP-tag and SNAP-tag can be used for orthogonal and complementary labeling of two proteins simultaneously in the same cells. 


    There are two steps to using this system: subcloning and expression of the protein of interest as a CLIP-tag fusion, and labeling of the fusion with the CLIP-tag substrate of choice. The use of BTC during the labeling of fusion proteins with CLIP-Cell substrates is described in this document.

    Figure 1: Structure of CLIP-Cell Block (MW 286.1)

    Properties and Usage

    Materials Required but not Supplied

    • Cells expressing CLIP-tag fusion proteins 
    • Tissue culture materials and media 
    • Transfection reagents 
    • Fluorescence microscope with suitable filter set 
    • DMSO

    Storage Temperature

    -20°C

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Cellular Protein Labeling (Blocking Assay):
      The blocking compound is pre-incubated with cells  expressing the target protein followed by labeling of the target protein.  No labeling is detected by fluorescence microscopy
    • In Vitro Protein Labeling:

      The product is tested in an in vitro protein labeling reaction. After incubation the labeled product is visualized on SDS-PAGE by fluorescent detection and verified by mass spectrometry.

    • Physical Purity (HPLC):
      The purity of the product is determined by HPLC analysis.

    Notes

    1. Storage: CLIP-Cell Block should be stored at -20°C (long term) or at 4°C (short term). With proper storage at -20°C, CLIP-Cell Block is stable for at least three years dry or for three months when dissolved in DMSO.
    2. For troubleshooting please refer to the instructions supplied with CLIP-Cell products as appropriate.
    3. Please note that there is a constant turnover and resynthesis of proteins in the cell. After having blocked all existing CLIP-tag fusion proteins within the cell, new CLIP-tag fusion protein molecules may be synthesized in the meantime and may get labeled during incubation with a fluorescent CLIP-tag substrate. This will give the impression that the blocking was ineffective. In order to minimize these effects of protein synthesis and protein transport, cells may have to be treated with cycloheximide and incubation with the fluorescent CLIP-tag substrate may have to be performed at 4°C.

    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.

    Datacards

    The Product Summary Sheet, or Data Card, 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 the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    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. Can cells expressing SNAP-tag be fixed prior to labeling?
    14. Can SNAP-tag be multiplexed with other protein labeling systems (GFP, Antibody)?
    15. Can you use SNAP-tag for in vivo FRET?
    16. Can cell-impermeable substrates be microinjected into cells, and how is the excess substrate exported?
    17. Does the SNAP-tag labeling reaction work in yeast?
    18. What happens to the fluorophore upon proteolysis?
    19. What conditions are recommended for SNAP-tag labeling in vitro?
    20. What conditions are incompatible with SNAP-tag labeling in vitro?
    21. Can SNAP-tag fusion proteins be labeled in a cell lysate?
    22. I have a compound that I would like to couple to a BG derivative. Where can I get advice?
    23. What is the difference between SNAP-tag and ACP-tag?
    24. Cellular Imaging and Analysis FAQs
    1. Use with CLIP-tag substrates (S9220)
    2. Labeling of Proteins in vitro (S9220)

    Selection Tools

    Usage Guidelines & Tips

    Troubleshooting Guides

    After addition of DMSO, pipette up and down at least 10-20 times and vortex vigorously for at least one full minute to ensure full dissolution of the substrate.

    After diluting the substrate in complete medium, thoroughly pipette up and down at least 10 times to help reduce background.

    Increasing substrate concentration and/or reaction time usually results in higher background and does not necessarily increase the signal-to-background ratio (SNR).