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  • CoA-Biotin

    Description

    CoA Biotin is a non cell-permeable substrate based on biotin with an amidocaproyl linker. It is suitable for applications such as biotinylation of ACP-tag or MCP-tag fusion proteins on the surface of living cells for detection with streptavidin fluorophore conjugates or labeling in solution for analysis by SDS-PAGE/Western Blot or for capture with streptavidin for binding and interaction studies. This package contains 50 nmol of CoA Biotin substrate, sufficient to make 10 ml of a 5 μM solution for the labeling of ACP-tag or MCP-tag fusion proteins on cells. 

    The ACP-tag and MCP-tag are small protein tags (8 kDa) based on the acyl carrier protein. MCP-tag contains two mutations (D36T and D39G). Both allow the specific, covalent attachment of virtually any molecule to a protein of interest. 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 or the MCP-tag by a phosphopantetheinyl 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) will preferentially label the ACP-tag, SFP Synthase (NEB #P9302) will label both ACP-tag and MCP-tag. 

    There are two steps to using this system: subcloning and expression of the protein of interest as an ACP-tag or MCP-tag fusion, and labeling of the fusion protein using the appropriate synthase with the CoA substrate of choice. Expression of ACP- and MCP-tagged proteins is described in the documentation supplied with the pACP-tag(m)-2 (NEB #N9322) and pMCP-tag(m) (NEB #N9317) vectors, respectively. The labeling of the fusion proteins with the CoA Biotin is described below.
    S9351a
    Figure 1. Structure of CoA-Biotin (MW 1365.3 g/mol)

    Properties and Usage

    Materials Required but not Supplied

    • ACP Synthase (NEB #P9301) for labeling ACP-tag 
    • SFP Synthase (NEB #P9302) for labeling ACP-tag or MCP-tag 
    • Cells expressing ACP-tag or MCP-tag fusion proteins 
    • Tissue culture materials and media 
    • Transfection reagents 
    • Fluorescence microscope with suitable filter set 
    • DMSO

    Storage Temperature

    -20°C

    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.
    • 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.

    • Physical Purity (HPLC):
      The purity of the product is determined by HPLC analysis.

    Notes

    1. Storage: CoA Biotin should be stored at -20°C (long term) or at 4°C in the dark (short term, less than 4 weeks). Protect the substrate from light and moisture. With proper storage at -20°C the substrate 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.

    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. Labeling on the Surface of Cells (S9351)
    2. Labeling of Proteins in vitro (S9351)

    Selection Tools

    Troubleshooting Guides

    Application Notes

    After addition of DMSO, pipette up and down at least 10-20 times and vortex vigorously for at least one full minute to ensure full dissolution of the substrate.

    After diluting the substrate in complete medium, thoroughly pipette up and down at least 10 times to help reduce background.

    Increasing substrate concentration and/or reaction time usually results in higher background and does not necessarily increase the signal-to-background ratio (SNR).