激发光660 nm 发射光 673 nm. 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.
细胞非通透性底物（CoABiotin）通过一个氨己酰基与生物素相连。生物素标记物可用于对活细胞内部或表面的融合蛋白的生物素化，从而检测链亲和素荧光基耦联物，或在溶液中标记，以进行 SDS-PAGE/ Western blot 分析。生物素标记物也被用于与链霉亲和素结合，以进行结合和相互作用方面的研究。
ACP 合成酶 (4' -磷酸泛酰巯基乙胺基转移酶) 催化辅酶 A (CoA) 上的取代基共价转移至暴露于活细胞表面的 ACP-tag 融合蛋白。ACP Synthase (4´-phosphopantetheinyl transferase) catalyzes the covalent transfer of substituents from derivatized coenzyme A (CoA) to ACP-tagged fusion proteins exposed on the surface of living cells. The 25 nmoles of ACP Synthase provided is sufficient to make 25 ml of a 1 µM ACP-tag fusion protein labeling solution.
The ACP-tag and MCP-tag are small tags (8 kDa) based on the acyl carrier protein (ACP). MCP-tag contains two mutations (D36-T36 and D39-G39). Both allow the specific, covalent attachment of virtually any molecule to a protein of interest. Substrates for labeling are derivatives of coenzyme A (CoA). In the labeling reaction, the substituted phosphopantetheine group of CoA is covalently attached to a conserved serine residue on the ACP-tag or the MCP-tag by a phosphopantetheine 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 will preferentially modify the ACP-tag, SFP Synthase (NEB #P9302 ) will modify both ACP-tag and MCP-tag. This principle can be employed for sequential dual labeling of two different proteins that localize to the cell surface. Cells co-expressing one ACP-tag fusion protein and one MCP-tag fusion protein can be incubated with ACP Synthase and one CoA substrate followed by labeling with SFP Synthase and a different CoA substrate.
There are two steps to using this system: cloning and expression of the protein of interest as an ACP-tag fusion, and labeling of the fusion protein using the ACP Synthase with the CoA substrate of choice. In this document, the labeling of fusion proteins with CoA substrates is described. The cloning of ACP-tag protein fusions is described in the documentation supplied with the ACP-tag plasmids.
激发光506 nm 发射光 526 nm. ACP/MCP-tag的标记基团与辅酶 A（CoA）的磷酸泛酰巯基乙胺耦联，在 ACP 或 SFP 合成酶作用下，完成标记反应。标记反应仅特异地发生在细胞表面表达的融合蛋白上。虽然 ACP- 和 MCP-tag 使用相同底物，但是，反应特异性却不同，表现在需要用不同的合成酶进行标记反应。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 #P9301) will preferentially label 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.
激发光554 nm 发射光 568 nm. ACP/MCP-tag的标记基团与辅酶 A（CoA）的磷酸泛酰巯基乙胺耦联，在 ACP 或 SFP 合成酶作用下，完成标记反应。标记反应仅特异地发生在细胞表面表达的融合蛋白上。虽然 ACP- 和 MCP-tag 使用相同底物，但是，反应特异性却不同，表现在需要用不同的合成酶进行标记反应。CoA 547 is a photostable fluorescent substrate that can be used to label ACP-tag and MCP-tag fusion proteins exposed on the surface of living cells. This cell-impermeable substrate is based on the Dyomics dye DY-547, and is suitable for standard TAMRA and Cy3 filter sets. It has an excitation maximum at 554 nm and an emission maximum at 568 nm. This package contains 50 nmol of CoA 547 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 label 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.
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