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  • SNAP-Cell® 360

    Description

    SNAP-Cell® 360 is a blue fluorescent substrate that can be used to label SNAP-tag® fusion proteins inside living cells, on cell surfaces, or in vitro. This cell-permeable substrate (CP-360) is based on aminomethylcoumarin and is suitable for DAPI filter sets or appropriate blue lasers. It has an excitation maximum at 357 nm and an emission maximum at 437 nm. This package contains 50 nmol of CP-360 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 dye-substituted benzyl group of the substrate is covalently attached to the SNAP-tag.

    There are two steps to using this system: subcloning 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 the 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 360 (bright blue) for 30 minutes.
    Figure 2:
    Excitation (dotted line) and emission spectra of SNAP-Cell-360 coupled to SNAP-tag in buffer at pH 7.5
    S9101b_thumb
    Figure 3:  Structure of SNAP-Cell-360 (MW 479.9)

    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

    437nm

    Excitation

    357nm

    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. While the majority of SNAP-tag substrates are benzylguanine derivatives, the SNAP-tag is also able to recognize chloropyrimidine derivatives. SNAP-Cell 360 is one such example.
    2. Storage: SNAP-Cell 360 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 (S9101)
    2. Labeling of Proteins in vitro (S9101)
    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 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.

    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).