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).
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 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.
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
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