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  • pACP-ADRβ2 Control Plasmid

    Discontinued Date

    01/01/2013
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    Categories:
    Discontinued Products

    Description

    This control plasmid contains the gene encoding the Beta-2 Adrenergic Receptor cloned as a fusion to the C-terminus of the ACP-tag. The signal peptide fused to the N-terminus of ACP-tag is based on the 5HT3A Serotonin Receptor. The Beta-2 Adrenergic Receptor is a member of the G protein coupled receptors and mediates the catecholamine-induced activation of adenylate cyclase through the action of G proteins.

    The ACP-Beta-2 Adrenergic Receptor is inserted in the plasma membrane with the ACP-tag exposed to the extracellular side of the membrane. When labeled with CoA substrates, it gives a selective cell membrane fluorescence labeling pattern.The full sequence and map for pACP-ADRβ2 can be downloaded here.

    The ACP-tag is a small protein tag (8 kDa) based on the acyl carrier protein (ACP). It allows the specific, covalent attachment of virtually any molecule to a protein of interest. ACP-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 ACP-tag by a phosphopantetheine transferase (ACP or SFP Synthase). Having no cysteines, the ACP-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: subcloning and expression of the protein of interest as an ACP-tag fusion, and labeling of the fusion protein with the CoA substrate of choice. The cloning and expression of ACP-tag protein fusions is described in documents provided with the pACP-tag(m)-2 cloning plasmid. The labeling of fusion proteins with CoA substrates is described in the documentation supplied with CoA substrates and ACP or SFP Synthase.

    Live U-2 0S cells transiently transfected with pACP-ADRβ2. Cells were labeled with CoA 547 using SFP Synthase for 30 minutes and imaged by confocal microscopy.


    N9321b

    Properties and Usage

    Materials Required but not Supplied

    • Mammalian cell lines 
    • Transfection reagents 
    • CoA substrates 
    • ACP or SFP Synthase 
    • Tissue culture reagents and media

    Storage Temperature

    -20°C

    Notes

    1. Storage: pACP-ADRβ2 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.

    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.
    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 ACP-ADRβ2 Fusions (N9321)

    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.