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  • CLIP-Surface™ 547

    Description

    CLIP-Surface™ 547 is a photostable red fluorescent substrate that can be used to label CLIP-tag™ fusion proteins on the surface of living cells or in vitro. This cell impermeable substrate (BC-547) is based on the Dyomics dye DY-547 and is suitable for use with standard TAMRA or Cy3 filter sets. It has an excitation maximum at 554 nm and emission maximum at 568 nm. This package includes 50 nmol of CLIP-Surface 547 substrate, sufficient to make 10 ml of a 5 µM CLIP-tag fusion protein labeling solution.

    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-DNA-alkyltransferase (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 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. Expression of CLIP-tag fusion proteins is described in the documentation supplied with CLIP-tag plasmids. The labeling of the fusion proteins on the cell surface with the CLIP-tag substrate is described in this document.

    Figure 1:
    Live CHO-K1 cells transiently transfected with pCLIP-NK1R. Cells were labeled with CLIP-Surface 547 (red) for 60 minutes and counterstained with Hoechst 33342 (blue).
    Figure 2:
    Excitation (dotted line) and emission (full line) spectra of CLIP-Surface 547 coupled to CLIP-tag in buffer at pH 7.5.
    S9233b_thumb
    Figure 3. Structure of CLIP-Surface 547 (MW 851.0 g/mol)

    Properties and Usage

    Materials Required but not Supplied

    • Cells expressing CLIP-fusion proteins. The protein of interest can be expressed with the CLIP-tag as either an N- or a C-terminal fusion, but note that the tag needs to be exposed to the extracellular surface of the plasma membrane for labeling with CLIP-Surface 547.
    • Tissue culture materials and media
    • Transfection reagents
    • Fluorescence microscope with suitable filter set
    • DMSO

    Emission

    568nm

    Excitation

    554nm

    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 (Cell Surface):
      The product is tested on cells expressing the target protein on the surface.  The surface target is labeled and visualized 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.

    Notes

    1. Storage: CLIP-Surface 547 should be stored at -20°C (long term) or at 4°C in the dark (short term, less than 4 weeks). Protect the substrate from light and moisture. With proper storage at -20°C the substrate should be stable for at least three years dry or 3 months dissolved in DMSO.

    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. Cellular Imaging and Analysis FAQs
    1. Cellular Labeling (S9233)
    2. Labeling of Proteins in vitro (S9233)
    3. View the video "Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging" in the Journal of Visualized Experiments (JoVE)

    Selection Tools

    Troubleshooting Guides

    Application Notes

    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).