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  • ACP-Surface Starter Kit

    Description

    The ACP-tag is a novel tool for the specific, covalent attachment of virtually any molecule to a cell surface protein of interest, providing simplicity and extraordinary versatility to the imaging of proteins in live and fixed cells and to the study of proteins in vitro. The creation of a single gene construct yields a tagged fusion protein capable of covalent derivatization with a variety of functional groups, including fluorophores and biotin. This system provides a powerful and unique tool to study the role of cell surface proteins in a variety of highly dynamic processes, including receptor internalization, turnover and complex formation.

    The ACP-tag and the related MCP-tag are small protein tags (77 amino acids, 8 kDa) based on the acyl carrier protein from E. coli. Both can be enzymatically modified with fluorophores, biotin etc. using substrates that are derivatives of coenzyme A (CoA). In the labeling reaction, the substituted phosphopantetheine group of the CoA substrate is covalently attached to a conserved serine residue of the ACP-tag or the MCP-tag by a 4´-phosphopantetheinyl transferase (SFP Synthase from B. subtilis, or ACP Synthase from E. coli) (Figure 1). The ACP-tag has a number of features that make it ideal for a variety of protein labeling applications. The rate of the reaction of the synthase with CoA derivatives is largely independent of the nature of the synthetic probe attached to CoA, permitting the labeling of ACP and MCP fusion proteins with a wide variety of functional groups (Figure 2). The ability to turn on the signal at will allows time-resolved analysis of protein trafficking and receptor internalization. Having no cysteines, the ACP-tag and the MCP-tag are particularly suited for specifically labeling cell-surface proteins, and should be useful for labeling secreted proteins with disulfide bridges such as antibodies. Finally, the availability of orthogonal protein labeling systems from NEB permits simultaneous labeling of multiple proteins in a single cell (SNAP-tag, a variant of the human DNA repair enzyme hAGT that transfers a label onto itself from O6-benzylguanine substrates, and CLIP-tag, a SNAP-tag variant that transfers a label onto itself from O2-benzylcytosine substrates). 

    While the ACP Synthase (NEB #P9301) will modify predominantly the ACP-tag, the included SFP Synthase will efficiently modify both the ACP-tag and MCP-tag (the MCP-tag contains two mutations relative to the ACP-tag, D36T and D39G, which abolish recognition by the ACP Synthase while preserving recognition by SFP Synthase). This principle can be employed for sequential dual labeling of two different proteins that localize to the cell surface. Cells co-expressing one ACP-tag fusion protein and one MCP-tag fusion protein are first incubated with ACP Synthase and one CoA substrate to label the ACP-tag, followed by incubation with the SFP Synthase and a different CoA substrate to label the MCP-tag.

    The ACP-Surface Starter Kit contains a mammalian expression plasmid (pACP-tag(m)-2) encoding the ACP-tag flanked by restriction sites for cloning a gene of interest, SFP Synthase, 1 M MgCl2 and two non-cell-permeable fluorescent CoA substrates. A positive control plasmid (pACP-ADRβ2), encoding an ACP-tagged protein (Beta-2 Adrenergic Receptor) with a well-characterized cell surface localization, is also included. There are two steps to using this system: subcloning and expression of the protein of interest as an ACP-tag fusion, and enzymatically labeling the fusion with the CoA substrate of choice.

    Figure 1:
    ACP-tag labeling reaction.
    Figure 2:  Live cell imaging of ACP-tag fusion proteins.
    Live CHO-K1 cells transiently transfected with pACP-GPI. Cells were labeled with CoA 488 (green) in the presence of ACP Synthase for 30 minutes.
    Figure 3:  Live cell imaging of ACP-tag fusion proteins.
    Live CHO-K1 cells transiently transfected with pACP-GPI. Cells were labeled with CoA 547 (red) in the presence of SFP Synthase for 30 minutes.

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    pACP-ADRβ2 Control Plasmid-200.5 mg/ml
    pACP-tag(m)-2 Vector-200.5 mg/ml
    SFP Synthase-2040 μM
    CoA 547-2010 nmol
    CoA 488-2010 nmol
    1M MgCl2100 mM

    Properties and Usage

    Materials Required but not Supplied

    • Mammalian Cell Lines 
    • DNA Transfection Reagents 
    • Standard Tissue Culture Media and Plasticware 
    • DMSO 
    • Hoechst 33342 for Nuclear Staining (optional)

    Notes

    1. For long-term storage, all kit components should be stored at -20˚C. Plasmid solutions can be stored at 4˚C for up to one week. With proper storage at -20°C the SFP Synthase should be stable for at least two years, and the CoA substrates should be stable for at least three years dry or 3 months dissolved in DMSO.

    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.

    Manuals

    The Product Manual 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 these document files: manual[Catalog Number].
    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. Are ACP-tag substrates stable to fixation?
    6. Can ACP-tag be multiplexed with other protein labeling systems (GFP, Antibody)?
    7. Can you use ACP-tag for in vivo FRET?
    8. Does the ACP-tag labeling reaction work in Yeast?
    9. Can I clone my protein as fusion to the N- or C-terminus of the ACP-tag?
    1. Cell Surface Labeling ACP-Surface Starter Kit
    2. Labeling of Proteins in vitro for ACP-Surface Starter Kit

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