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  • SNAP-Cell® Oregon Green®

    Description

    SNAP-Cell Oregon Green is a photostable green fluorescent substrate that can be used to label SNAP-tag® fusion proteins inside living cells. This cell-permeable substrate (BG-Oregon Green) is based on the Invitrogen dye, Oregon Green and is suitable for standard fluorescein filter sets. It has an excitation maximum at 490 nm and an emission maximum at 514 nm. Conjugates of Oregon Green are more photostable than those of fluorescein, and their fluorescence properties are essentially pH insensitive in the physiological pH range. This package contains 50 nmol of SNAP-Cell Oregon Green substrate, sufficient to make 10 ml of a 5 µM SNAP-tag fusion protein labeling solution.

    The SNAP-tag is a novel tool for protein research, allowing the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is based on mammalian O6-alkylguanine-DNA-alkyltransferase (AGT). SNAP-tag substrates are derivatives of benzylguanines and benzylchloropyrimidines. In the labeling reaction, the dye-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:
    Live U2OS cells transiently transfected with pSNAP-Cox8A (mitochondrial expression). Cells were labeled with SNAP-Cell Oregon Green (green) for 15 minutes and counterstained with Hoechst 33342 (blue).
    Figure 2:
    Excitation (dotted line) and emission spectra after coupling of SNAP-Cell Oregon Green to SNAP-tag in buffer at pH 7.5.

    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

    514nm

    Excitation

    490nm

    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 (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.

    Notes

    1. Storage:
      SNAP-Cell Oregon Green 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.
    2. SNAP-Cell Oregon Green is not suitable for labeling of cell surface SNAP-tag fusion proteins. The Oregon Green dye is based on a fluorinated fluorescein modified with pivaloyl groups which is uncharged and essentially non-fluorescent. It becomes fluorescent once inside the cell where it is hydrolyzed by non-specific esterases.

    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 (S9104)
    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 (S9104)

    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).

    After the wash steps, incubate the intracellular labeling samples at 37°C, 5% CO2 for 30 extra minutes to allow any unincorporated fluorophore to diffuse out of the cells.