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    pACP-tag(m)-2 Vector

    Discontinued Date

    01/05/2015
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    Categories:
    Discontinued Products

    Description

    pACP-tag(m)-2 Vector is a mammalian expression plasmid encoding ACPwt, an ACP-tag protein, which is expressed under control of the CMV promoter. The expression plasmid has an IRES (Internal Ribosome Entry Site) and a neomycin resistance gene downstream of the ACP-tag for the efficient selection of stable transfectants. This plasmid is intended for the cloning and transient or stable expression of ACP-tag protein fusions in mammalian cells. pACP-tag(m)-2 contains two multiple cloning sites to allow cloning of the fusion partner as a fusion to the C-terminus of the ACP-tag and an appropriate signal peptide to the N-terminus of the ACP-tag.

    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. In this document, the cloning and expression of ACP-tag protein fusions is described. The labeling of fusion proteins with CoA substrates is described in the documentation supplied with CoA substrates and ACP or SFP Synthase.



    Cloning Region of pACP-tag(m)-2
    Unique restriction sites in the regions flanking the ACP-tag gene are displayed above the coding strand. The complete sequence for pACP-tag(m)-2 and pACP-ADRβ2 can be downloaded here 

    DNASU is a central repository for plasmid clones and collections that may also be helpful.

    Advantages and Features

    Features


    BACKGROUND:

    A plasmid map and the sequence of the cloning region can be found above. The complete plasmid sequence can be downloaded. This plasmid encodes the gene ACPwt which is the wild type Acyl Carrier Protein from E. coli. In the plasmid sequence, the ACPwt gene is encoded from bp 975 to 1208. The ACPwt gene was cloned via EcoRI and SbfI including additional flanking restriction sites.

    This plasmid is intended for the cloning and stable or transient expression of ACP-tag protein fusions in mammalian cells. It is suitable for the efficient production of stable cell lines expressing ACP-tag gene fusions. The plasmid contains the CMV promoter followed by the genes for the ACP-tag and neomycin resistance separated by the IRES of the encephalomyocarditis virus (ECMV), which permits the translation of two open reading frames from one messenger RNA; therefore after selection of stable mammalian cells for neomycin resistance, nearly all surviving colonies should stably express the ACP-tag fusion protein. Unless the expression experiments require a pure population of cells, the pool of resistant cells can simply be used, otherwise cell clones can be isolated and characterized using standard procedures.

    The plasmid contains the β-lactamase (Ampicillin resistance) gene for maintenance in bacteria. The gene of interest should be cloned downstream of the ACP-tag coding sequence, as a fusion to the C-terminus of the ACP-tag. An appropriate cell surface signal peptide should be cloned upstream of the ACP-tag as an N-terminal fusion. A Kozak sequence is located upstream of the ACPwt gene to increase the translation efficiency of the fusion protein. The ACPwt gene can be isolated from the plasmid using PCR or direct cloning in order to subclone it into a different vector of choice.

    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-tag(m)-2 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.

    FAQs

    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?

    Protocols

    1. Cloning of ACP-tag Fusions in pACP-tag(m)-2 (N9322)
    2. Expression of ACP-tag Fusions (N9322)

    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.

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    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.