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  • pMCP-GPI Control Plasmid

    Discontinued Date

    01/01/2013
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    Categories:
    Discontinued Products

    Description

    This control plasmid contains a glycosylphosphatidylinositol (GPI) anchor sequence cloned upstream of the MCP-tag coding sequence in pMCP-tag(m). This sequence directs the fusion protein to the plasma membrane with the MCP-tag exposed on the outside surface, giving a localized control signal when labeled with CoA substrates. GPI is present in all eukaryotic cells where it anchors glycoproteins to the cell surface. The GPI anchor helps increase the mobility of membrane proteins thereby supporting signal transduction and cellular transport. In addition, the GPI anchor plays an important role in creating antigens on cell surfaces. 

    The MCP-tag is a small protein tag (8 kDa) based on the acyl carrier protein (ACP) containing two mutations (D36-T36 and D39-G39), for labeling cell membrane proteins. It allows the specific, covalent attachment of virtually any molecule to a protein of interest. MCP-tag substrates are derivatives of coenzyme A (CoA). In the labeling reaction, the substituted phosphopantetheine group of CoA is covalently attached to a conserved serine residue of the MCP-tag by a phosphopantetheine transferase (SFP Synthase). Having no cysteines, the MCP-tag is particularly suited for specifically labeling cell surface proteins, and should be useful for labeling secreted proteins with disulfide bridges such as antibodies.

    There are two steps to using this system: sub cloning and expression of the protein of interest as an MCP-tag fusion, and labeling of the fusion protein using SFP Synthase with the substrate of choice.The cloning and expression of MCP-tag protein fusions is described in documents provided with the pMCP-tag(m) cloning plasmid. The labeling of fusion proteins with CoA substrates is described in the documentation supplied with CoA substrates and SFP Synthase.

    Live CHO-K1 cells transiently transfected with pMCP-GPI. Cells were labeled with CoA 488 (green) using SFP Synthase for 30 minutes.


    N9320_pMCP-GPI

    Properties and Usage

    Materials Required but not Supplied

    • Mammalian cell lines 
    • Transfection reagents 
    • CoA substrates
    • SFP Synthase

    Storage Temperature

    -20°C

    Notes

    1. pMCP-GPI is supplied in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) at a concentration of 0.5 µg/µl. Plasmid solutions can be stored at 4°C for up to one week. For long-term storage -20°C is recommended.
    2. NEB 10-beta Competent E. coli (High Efficiency) (NEB #C3019) is recommended for propagating this control plasmid.

    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. What is the ACP-tag?
    2. How does it work?
    3. How specific is the binding of substrate to the ACP-tag?
    4. What linker type and length would you recommend?
    5. Can I clone my protein as fusion to the N- or C-terminus of the ACP-tag?
    6. Are ACP-tag substrates stable to fixation?
    7. Can ACP-tag be multiplexed with other protein labeling systems (GFP, Antibody)?
    8. Can you use ACP-tag for in vivo FRET?
    9. Does the ACP-tag labeling reaction work in Yeast?
    1. Expression of MCP-GPI (N9320)

    Selection Tools

    Troubleshooting Guides

    Interactive Tools

    If you generate more plasmid DNA by bacterial transformation, we recommend isolating the plasmid DNA using an endotoxin-free plasmid prep kit prior to transfection into mammalian cells.