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  • SNAP-Cell® Fluorescein

    Description

    SNAP-Cell® Fluorescein is a green fluorescent substrate that can be used to label SNAP-tag® fusion proteins inside living cells or in vitro. This cell-permeable substrate (BG-Fluorescein) is based on diacetylfluorescein and is suitable for standard fluorescein filter sets. Diacetylfuorescein is essentially non-fluorescent, but it becomes fluorescent inside the cell when it is hydrolyzed by non-specific esterases, yielding fluorescein. It has an excitation maximum at 500 nm and an emission maximum at 532 nm. This substrate has limited photostability. If this presents a problem, we recommend using SNAP-Cell 505, which has similar spectral characteristics but much greater photostability. This package contains 50 nmol of SNAP-Cell Fluorescein substrate, sufficient to make 10 ml of a 5 µM SNAP-tag fusion protein labeling solution. 

    The SNAP-tag protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is a small protein based on mammalian O6-alkylguanine-DNA-alkyltransferase (AGT). SNAP-tag substrates are derivatives of benzylpurines and benzylchloropyrimidines, In the labeling reaction, the substituted benzyl group of the substrate becomes covalently attached to the SNAP-tag.

    There are two steps to using this system: sub-cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. Expression of SNAP-tag fusion proteins is described in the documentation supplied with SNAP-tag plasmids. The labeling of fusion proteins with the SNAP-tag substrate is described below.

    Figure 1:
    Live CHO-K1 cells transiently transfected with pSNAP-H2B. Cells were labeled with SNAP-Cell Fluorescein (green) for 15 minutes.
    Figure 3:
    Excitation (dotted line) and emission spectra of SNAP-Cell Fluorescein (fluorescent product of SNAP-Cell Fluorescein in cells) coupled to SNAP-tag in buffer at pH 7.5)
    S9107a_TN
    Figure 3:  Structure of SNAP-Cell Fluorescein (MW 712.7g/mol)

    Properties and Usage

    Materials Required but not Supplied

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

    Emission

    532nm

    Excitation

    500nm

    Storage Temperature

    -20°C

    Notes

    1. Storage:
      SNAP-Cell Fluorescein 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 3 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 (S9107)
    2. View the video "Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging" in the Journal of Visualized Experiments (JoVE)
    3. Labeling of Proteins in vitro (S9107)

    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).

    After the wash steps, incubate the intracellular labeling samples at 37°C, 5% CO2 for 30 extra minutes to allow any unincorporated fluorophore to diffuse out of the cells.