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  • SNAP-Cell® 505-Star

    Description

    SNAP-Cell® 505-Star is a photostable green 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-6-505) is based on the single isomer 6-carboxyrhodamine 110 and is suitable for standard fluorescein filter sets. It has an excitation maximum at 504 nm and an emission maximum at 532 nm. This package contains 50 nmol of SNAP-Cell 505-Star 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: 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 U-2 OS cells stably transfected with pSNAPf-H2B. Cells were labeled with SNAP-Cell 505-Star (green) for 15 minutes at 37°C, 5% CO2.
    Figure 2:
    Excitation (dotted line) and emission spectra of SNAP-Cell 505-Star coupled to SNAP-tag in buffer at pH 7.5
    S9103c
    Figure 3.
    Structure of SNAP-Cell 505-Star (MW 621.0 g/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

    504nm

    Notes

    1. Storage: SNAP-Cell 505-Star 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.

    FAQs

    1. Cellular Imaging and Analysis FAQs

    Tech Tips

    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).

    Protocols

    1. Cellular Labeling (S9103)
    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 (S9103)

    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.

    Selection Tools

    Troubleshooting Guides

    Application Notes

    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.

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

    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.