CoA 488 is a photostable fluorescent substrate used to label ACP-tag and MCP-tag fusion proteins exposed on the surface of living cells. This cell-impermeable CoA substrate is based on the ATTO-TEC dye ATTO 488, and is suitable for standard fluorescein filter sets. It has an excitation maximum at 506 nm and an emission maximum at 526 nm. This package contains 50 nmol of CoA 488 substrate, 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 #9301) will preferentially modify the ACP-tag, SFP Synthase (NEB #P9302) will modify 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 pACP-tag and pMCP-tag plasmids, respectively. The labeling of the fusion proteins with the CoA substrate is described below.
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
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