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  • CoA 647

    Description

    CoA 647 is a photostable fluorescent substrate that can be used to label ACP-tag or MCP-tag fusion proteins exposed on the surface of living cells. This cell-impermeable substrate is based on the Dyomics dye DY-647P1, and is suitable for Cy5 lasers. It has an excitation maximum at 660 nm and an emission maximum at 673 nm. The 50 nmol of CoA 647 in each vial is sufficient to make 10 ml of a 5 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 (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).

    While ACP Synthase (NEB #P9301) will preferentially label the ACP-tag, SFP Synthase (NEB #P9302) will label both ACP-tag and MCP-tag.

    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.

    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 ACP-tag and MCP-tag plasmids, respectively. The labeling of the fusion proteins with the CoA substrate is described below. 

    Figure 1:
    Live U2OS cells transiently transfected with pACP-ADRB2. Cells were labeled with 5 μM CoA 647 (red) in the presence of ACP Synthase for 60 minutes and counterstained with Hoechst 33342 (blue).
    Figure 2:
    Excitation (dotted line) and emission spectra of CoA 647 coupled to ACP-tag in buffer at pH 7.4.
    S9350b
    Figure 3. Structure of CoA 647 (MW 1576.5 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 and 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

    Emission

    673nm

    Excitation

    660nm

    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.
    • Cellular Protein Labeling (Cell Surface):
      The product is tested on cells expressing the target protein on the surface.  The surface target is labeled and visualized 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. Optimizing Labeling: Optimal substrate concentrations and reaction times range from 1–10 µM and 30–60 minutes, respectively, depending on experimental conditions and expression levels of the ACP-tag or MCP-tag fusion protein. Best results are usually obtained at concentrations between 1 and 5 µM substrate and 60 minutes reaction time. Increasing substrate concentration and reaction time usually results in a higher background and does not necessarily increase the signal to background ratio.
    2. Stability of Labeling: The turnover and internalization rates of the ACP-tag or MCP-tag fusion protein under investigation may vary widely depending on the fusion partner. Where protein turnover is rapid, we recommend analyzing the cells under the microscope immediately after the labeling reaction or fixing the cells directly after labeling.
    3. Fixation of Cells: After labeling the ACP-tag or MCP-tag fusion proteins, the cells can be fixed with standard fixation methods such as para-formaldehyde, ethanol, methanol, methanol/acetone etc., without loss of signal. We are not aware of any incompatibility of the CoA label with any fixation method.
    4. Counterstaining: Cells can be counterstained with any live-cell dye that is compatible with the fluorescent properties of the substrate for simultaneous microscopic detection. We routinely add 5 µM Hoechst 33342 to the labeling medium as a DNA counterstain for nuclear visualization.
    5. Storage: CoA 647 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 3 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 of Proteins in vitro (S9350)
    2. Labeling on the Surface of Cells (S9350)

    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).