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  • SFP Synthase

    Description

    SFP Synthase (4´-phosphopantetheinyl transferase) catalyzes the covalent transfer of substituents from derivatized coenzyme A (CoA) to ACP- or MCP-tagged fusion proteins exposed on the surface of living cells. The 25 nmoles of SFP Synthase provided is sufficient to make 25 ml of a 1 µM ACP-tag or MCP-tag fusion protein labeling solution.

    The ACP-tag and MCP-tag are small protein tags (8 kDa) based on the acyl carrier protein. MCP-tag contains two mutations (D36-T36 and D39-G39). Both allow the specific, covalent attachment of virtually any molecule to a protein of interest. Substrates for labeling are derivatives of coenzyme A (CoA). In the labeling reaction, the substituted phosphopantetheine group of CoA is covalently attached to a conserved serine residue on the ACP-tag or the MCP-tag by a phosphopantetheine transferase (SFP Synthase or ACP Synthase). 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.

    While ACP Synthase (NEB #P9301) preferentially modify the ACP-tag, SFP Synthase preferentially modify the ACP-tag, SFP Synthase will modify both ACP-tag and MCP-tag. 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 can be incubated with ACP Synthase and one CoA substrate followed by labeling with SFP Synthase and a different CoA substrate.

    There are two steps to using this system: cloning and expression of the protein of interest as an ACP-tag or MCP-tag fusion, and labeling of the fusion protein using the SFP Synthase with the CoA substrate of choice. In this document, the labeling of fusion proteins with CoA substrates is described. The cloning of ACP-tag or MCP-tag protein fusions is described in the documentation supplied with the ACP-tag or the MCP-tag plasmids.

    Figure 1:
    SFP labeling reaction

    Product Source

    An E. coli strain carrying the cloned B.subtilis gene for 4´- phosphopantetheinyl transferase (SFP).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    1M MgCl2

    Properties and Usage

    Storage Temperature

    -20°C

    Storage Conditions

    50 mM HEPES
    150 mM NaCl
    1 mM DTT
    50% Glycerol
    pH 7.4

    Heat Inactivation

    No

    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 (Blocking Assay):
      The blocking compound is pre-incubated with cells  expressing the target protein followed by labeling of the target protein.  No labeling is detected 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: SFP Synthase is stable for at least 2 years when stored at -20°C.

    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. Instructions for Use with SFP Synthase (P9302)
    2. Labeling of Proteins in vitro (P9302)