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  • CLIP-Cell™ TMR-Star

    Description

    CLIP-Cell™ TMR-Star is a photostable red fluorescent substrate that can be used to label CLIP-tag™ fusion proteins inside living cells, on cell surfaces or in vitro. This cell-permeable substrate (BC-TMR) is based on tetramethylrhodamine and is suitable for standard rhodamine filter sets. It has an excitation maximum at 554 nm and emission maximum at 580 nm. This package includes 30 nmol of CLIP-Cell TMR-Star, sufficient to make 10 ml of a 3 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-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. Expression of CLIP-tag fusion proteins is described in the documentation supplied with CLIP-tag plasmids. The labeling of the fusion proteins with the CLIP-tag substrate is described in this document.

    Live COS-7 expressing pCLIP-H2B and pSNAP-ADRβ2 were labeled with CLIP-Cell TMR-Star (red) and SNAP-Alexa Fluor® 488 (green) for 60 minutes.

    Excitation (dotted line) and emission spectra of CLIP-Cell TMR-Star coupled to CLIP-tag in buffer at pH 7.5.
    S9219b_thumb
    Structure of CLIP-Cell TMR-Star (MW 642.7 g/mol)

    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

    Emission

    580nm

    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 (Intracellular):
      The product is tested on cells expressing the target protein intracellularly.  The intracellular target is labeled and visulaized 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-Cell TMR-Star 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. Labeling of Proteins in vitro (S9219)
    2. Cellular Labeling (S9219)
    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).