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-647, 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 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 ACP-tag and MCP-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 and MCP-tag
Cells expressing ACP-tag or MCP-tag fusion proteins
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