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ACP-Surface 启动试剂盒 :ACP-Surface Starter Kit 1 set

价格:¥

货号:E9300S
品牌:NEB

经销:基因有限公司

蛋白质标记系统启动试剂盒提供了标记ACP-tag 融合蛋白的所有必需组份。能在活细胞、固定细胞及体外将红色或绿色荧光基团共价连接到目标蛋白上。每种启动试剂盒包括一个编码所选 tag 的质粒和细胞非通透性的荧光标记物CoA 488和CoA 547。试剂盒中还提供一种阳性对照质粒,其编码有亚细胞定位明确的标签蛋白(如:膜蛋白、核蛋白等)。The ACP-tag is a novel tool for the specific, covalent attachment of virtually any molecule to a cell surface protein of interest, providing simplicity and extraordinary versatility to the imaging of proteins in live and fixed cells and to the study of proteins in vitro. The creation of a single gene construct yields a tagged fusion protein capable of covalent derivatization with a variety of functional groups, including fluorophores and biotin. This system provides a powerful and unique tool to study the role of cell surface proteins in a variety of highly dynamic processes, including receptor internalization, turnover and complex formation.

The ACP-tag and the related MCP-tag are small protein tags (77 amino acids, 8 kDa) based on the acyl carrier protein from E. coli. Both can be enzymatically modified with fluorophores, biotin etc. using substrates that are derivatives of coenzyme A (CoA). In the labeling reaction, the substituted phosphopantetheine group of the CoA substrate is covalently attached to a conserved serine residue of the ACP-tag or the MCP-tag by a 4´-phosphopantetheinyl transferase (SFP Synthase from B. subtilis, or ACP Synthase from E. coli) (Figure 1). The ACP-tag has a number of features that make it ideal for a variety of protein labeling applications. The rate of the reaction of the synthase with CoA derivatives is largely independent of the nature of the synthetic probe attached to CoA, permitting the labeling of ACP and MCP fusion proteins with a wide variety of functional groups (Figure 2). The ability to turn on the signal at will allows time-resolved analysis of protein trafficking and receptor internalization. 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. Finally, the availability of orthogonal protein labeling systems from NEB permits simultaneous labeling of multiple proteins in a single cell (SNAP-tag, a variant of the human DNA repair enzyme hAGT that transfers a label onto itself from O6-benzylguanine substrates, and CLIP-tag, a SNAP-tag variant that transfers a label onto itself from O2-benzylcytosine substrates).

While the ACP Synthase (NEB #P9301) will modify predominantly the ACP-tag, the included SFP Synthase will efficiently modify both the ACP-tag and MCP-tag (the MCP-tag contains two mutations relative to the ACP-tag, D36T and D39G, which abolish recognition by the ACP Synthase while preserving recognition by SFP Synthase). 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 are first incubated with ACP Synthase and one CoA substrate to label the ACP-tag, followed by incubation with the SFP Synthase and a different CoA substrate to label the MCP-tag.

The ACP-Surface Starter Kit contains a mammalian expression plasmid (pACP-tag(m)-2) encoding the ACP-tag flanked by restriction sites for cloning a gene of interest, SFP Synthase, 1 M MgCl2 and two non-cell-permeable fluorescent CoA substrates. A positive control plasmid (pACP-ADRβ2), encoding an ACP-tagged protein (Beta-2 Adrenergic Receptor) with a well-characterized cell surface localization, is also included. There are two steps to using this system: subcloning and expression of the protein of interest as an ACP-tag fusion, and enzymatically labeling the fusion with the CoA substrate of choice.

CoA-Biotin 50 nmol

价格:¥

货号:S9351S
品牌:NEB

经销:基因有限公司

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 :ACP Synthase 25 nmol

价格:¥

货号:P9301S
品牌:NEB

经销:基因有限公司

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.

SFP 合成酶 :SFP Synthase 25 nmol

价格:¥

货号:P9302S
品牌:NEB

经销:基因有限公司

SFP 合成酶 (4' -磷酸泛酰巯基乙胺基转移酶) 可以催化辅酶 A (CoA) 上的取代基共价连接到暴露于活细胞表面的 ACP- 或 MCP-tag 融合蛋白上。

CoA 488 50 nmol

价格:¥

货号:S9348S
品牌:NEB

经销:基因有限公司

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.

CoA 547 50 nmol

价格:¥

货号:S9349S
品牌:NEB

经销:基因有限公司

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.

CoA 647 50 nmol

价格:¥

货号:S9350S
品牌:NEB

经销:基因有限公司

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.

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