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    SNAP-Surface® 594

    Description

    SNAP-Surface® 594 is a photostable fluorescent substrate that can be used to label SNAP-tag® fusion proteins in solution or on the surface of living or fixed cells. This substrate (BG-594) is based on the ATTO-TEC dye ATTO 594 and is suitable for standard Texas Red and Alexa 594 filter sets. It has an excitation maximum at 606 nm and emission maximum at 626 nm. This package includes 50 nmol of SNAP-Surface 594 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 manmmalian 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: 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.
    Excitation (dotted line) and emission spectra of SNAP-Surface 594 coupled to SNAP-tag in buffer at pH 7.5

    Properties and Usage

    Materials Required but not Supplied

    • Cells expressing SNAP-tag Fusion proteins. Proteins of interest can be expressed with the SNAP-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 SNAP-Surface 594.
    • Tissue culture materials and media
    • Transfection reagents
    • Fluorescence microscope with suitable filter set
    • DMSO

    Emission

    626nm

    Excitation

    606nm

    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.

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

    Notes

    1. Storage: SNAP-Surface 594 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 (S9134)
    2. Labeling Proteins in vitro (S9134)
    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).