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  • SNAP-Vista® Green

    Description

    SNAP-Vista® Green is a green fluorescent substrate that can be used to label SNAP-tag® fusion proteins (in cell lysates or purified proteins) for detection by SDS-PAGE. This substrate (BG-Vista Green) is based on fluorescein and is optimized for excitation with the 488 nm laser excitation line in a laser based gel scanner. It can also be excited using 360 nm light from a standard UV-transilluminator. It has an excitation maximum at 500 nm and emission maxima at 524 nm. This package includes 50 nmol of SNAP-Vista Green substrate, sufficient to label one hundred 20 µl samples containing SNAP-tag fusion protein for in-gel detection. 

    The SNAP-tag protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is 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: 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 for detection by SDS-PAGE is described in this document.

    Figure 1:
    Excitation (dotted line) and emission spectra of SNAP-Vista Green coupled to SNAP-tag in buffer at pH 7.5.
    Figure 2:
    Typical SDS-PAGE gel from a gel scanner (Molecular Dynamics Storm Imager, 488 nm excitation).
    Lane 1 fluorescent MW marker 
    Lane 2-4 SNAP-His (22 kD) 0.1, 0.5, 1.0 µg 
    Lane 5-7 SNAP-CyA (40 kD) 0.1, 0.5, 1.0 µg 
    Lane 8-10 MBP-SNAP (63 kD) 0.1, 0.5, 1.0 µg
    9147a_thumb
    Figure 3: Structure of SNAP-Vista Green (MW 628.6 g/mol)

    Properties and Usage

    Emission

    524nm

    Excitation

    500nm

    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.
    • 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: Store substrate at -20°C. After dissolving SNAP-Vista Green store it in aliquots at -20°C in the dark for up to three months. Once an aliquot is thawed it can be stored at 4°C in the dark for up to two weeks. Make sure substrate, which may precipitate during freezing, is completely redissolved before use.
    2. With proper storage at -20°C, this substrate should be stable for at least three years dry, or three 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 Mammalian Cell Lysates (S9147)
    2. Labeling Proteins in vitro (S9147)

    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).