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  • SNAP-Surface® 425

    Discontinued Date

    01/01/2013
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    Categories:
    Discontinued Products

    Description

    SNAP-Surface® 425 is a photostable fluorescent substrate that can be used to label SNAP-tag® fusion proteins on cell surfaces or in solution. This substrate (BG-425) is based on the ATTO-TEC dye ATTO 425 and is suitable for appropriate blue lasers and filter sets. It has an excitation maximum at 438 nm and emission maximum at 489 nm. This package includes 50 nmol of SNAP-Surface 425 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 protein based on human O6-alkylguanine-DNA alkyltransferase (hAGT). SNAP-tag substrates are fluorophores, biotin or beads conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is 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 the fusion proteins with the SNAP-tag substrate is described below.

    Figure 1:
    Excitation (dotted line) and emission spectra of SNAP-Surface 425 coupled to SNAP-tag in buffer at pH 7.5
    S9126a_thumb
    Figure 2:  Structure of SNAP-Surface 425 (MW 725.8 g/mol)

    Properties and Usage

    Materials Required but not Supplied

    • Cells expressing SNAP-tag 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 425.
    • Tissue culture materials and media
    • Transfection reagents
    • Fluorescence microscope with suitable filter set
    • DMSO

    Emission

    489nm

    Excitation

    438nm

    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: SNAP-Surface 425 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.
    1. Cellular Imaging and Analysis FAQs
    1. Cellular Labeling (S9126)
    2. Labeling proteins in vitro (S9126)
    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).