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  • SNAP-Surface® Block

    Description

    SNAP-Surface® Block (C8 propanoic acid benzylguanine, CBG) is a nonfluorescent compound that blocks the reactivity of the SNAP-tag® in solution or on the surface of living cells. It can be used to generate inactive controls in live cell labeling experiments performed with SNAP-tag fusion proteins. SNAP-Surface Block reacts with the SNAP-tag irreversibly, inactivating it for subsequent labeling steps. This blocker is largely membrane impermeable essentially limiting blocking to cell surface-exposed SNAP-tags. 

    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 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 instructions supplied with SNAP-tag plasmids. The labeling of SNAP-tag fusion proteins with SNAP-Surface substrates is described in the instructions supplied with SNAP-Surface substrates. The use of SNAP-Surface Block during the labeling of fusion proteins with SNAP-Surface substrates is described below.
    Figure 1: Structure of SNAP-Surface Block (MW 313.31 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

    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 (Blocking Assay):
      The blocking compound is pre-incubated with cells  expressing the target protein followed by labeling of the target protein.  No labeling is detected 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 Block should be stored at -20°C (long term) or at 4°C (short term). With proper storage at -20°C, SNAP-Surface Block is stable for at least three years dry or 3 months when dissolved in DMSO.
    2. For troubleshooting please refer to the instructions supplied with SNAP-Surface products as appropriate.
    3. Please note that there is a constant turnover of proteins in the cell. Protein transport to the membrane and internalization followed by degradation or recycling, are constantly ongoing processes. After having blocked SNAP-tag on the cell membrane, newly synthesized protein may be transported to the cell surface and may get labeled during incubation with a fluorescent SNAP-tag substrate. This will give the impression that the blocking was ineffective. In order to minimize these effects of protein synthesis and protein transport, cells may have to be treated with cycloheximide and incubation with the fluorescent SNAP-tag substrate may have to be performed at 4°C.

    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. Use with SNAP-Surface substrates (S9143)
    2. Labeling of Proteins in vitro (S9143)

    Selection Tools

    Usage Guidelines & Tips

    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).