阻断剂是非荧光底物，在细胞内（SNAP-Cell Block）阻断 SNAP-tag 的反应。在 SNAP-tag 融合蛋白的活细胞标记实验中，可以用阻断剂制备无荧光对照。SNAP-Cell 阻断剂可顺利透过细胞膜，一旦进入细胞，即与 SNAP-tag 反应，使之在后续的标记反应中不可逆地失活。SNAP-Cell® Block (bromothenylpteridine, BTP) is a non-fluorescent compound that blocks the reactivity of the SNAP-tag® in solution or in living cells. It can be used to generate inactive controls in live cell labeling experiments performed with SNAP-tag fusion proteins. SNAP-Cell Block is highly membrane permeable and once in the cell reacts with the SNAP-tag, irreversibly inactivating it for subsequent labeling steps.
The SNAP-tag protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is a small protein based on mammalian O6-alkylguanine-DNA alkyltransferase (AGT). SNAP-tag substrates are derivatives of benzylpurines and benzylchloropyrimidines. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag.
There are two steps to using this system: sub-cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. Expression of SNAP-tag fusion proteins is described in the instructions supplied with SNAP-tag plasmids. The labeling of SNAP-tag fusion proteins with SNAP-Cell substrates is described in the instructions supplied with SNAP-Cell substrates. The use of SNAP-Cell Block during the labeling of fusion proteins with SNAP-Cell substrates is described below.
蛋白质标记系统启动试剂盒提供了标记 SNAP-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。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）。如果必要，试剂盒还会提供一个与目的标签相互作用的、非荧光的阴性对照阻断剂。The SNAP-Cell Starter Kit contains a mammalian expression plasmid (pSNAPf) encoding the SNAP-tag flanked by restriction sites for cloning a gene of interest, and two cell-permeable fluorescent SNAP-tag substrates. A positive control plasmid (pSNAPf-Cox8A), encoding a SNAP-tagged protein (cytochrome c oxidase) with a well-characterized mitochondrial localization, is also included. Lastly, a negative control “blocking agent” (SNAP-Cell Block) is included that interacts with the SNAP-tag, but is not fluorescent. There are two steps to using this system: subcloning and expression of the protein of interest as a SNAPf fusion, and labeling of the fusion with the SNAP-tag substrate of choice.
The SNAP-tag® is a novel tool for the specific, covalent attachment of virtually any molecule to a 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 forming a covalent linkage to a variety of functional groups, including fluorophores, biotin, or beads. This system provides a powerful and unique tool to study the role of proteins in a variety of highly dynamic processes, including protein trafficking, turnover, and complex formation.
The SNAP-tag is a 20 kDa mutant of the human DNA repair protein O6-alkylguanine- DNA alkyltransferase (hAGT) that reacts specifically and rapidly with benzylguanine (BG) and benzylchloropyrimidine (CP) derivatives, leading to covalent labeling of the SNAP-tag with a synthetic probe (Figure 1). The SNAP-tag has a number of features that make it ideal for a variety of protein labeling applications. The rate of the reaction of the SNAP-tag with these derivatives is largely independent of the nature of the synthetic probe attached to BG, permitting the labeling of SNAP fusion proteins with a wide variety of functional groups. Many of these SNAP-tag substrates are cell-permeable, allowing live-cell imaging of protein expression and localization (Figure 2). The ability to turn on the signal at will, together with the availability of a cell-permeable nonfluorescent blocking agent (SNAP-Cell® Block), allows time-resolved pulse-chase analysis of protein trafficking. Finally, the availability of orthogonal protein labeling systems from NEB permits simultaneous labeling of multiple proteins in a single cell (CLIP-tag™, a SNAP-tag variant that reacts exclusively with O2-benzylcytosine substrates, and the ACP/MCP tags, small protein tags which can be enzymatically labeled on the cell surface with Coenzyme A derivatives).
细胞通透性底物(SNAP-Cell）不仅能标记细胞内部也能标记细胞表面。激发光421nm，发射光444和484 nm。SNAP-Cell® 430 is a blue fluorescent substrate that can be used to label SNAP-tag® fusion proteins inside living cells, on cell surfaces, or in vitro. This cell-permeable substrate (BG-430) is based on diethylaminocoumarin and is suitable for appropriate blue lasers and filter sets. It has an excitation maximum at 421 nm and emission maxima at 444 and 484 nm. This package includes 50 nmol of SNAP-Cell 430 substrate, sufficient to make 10 ml of a 5 µM SNAP-tag fusion protein labeling solution.
The SNAP-tag protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA alkyltransferase (AGT). SNAP-tag substrates are derivatives of benzylpurines and benzylchloropyrimidines. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag.
There are two steps to using this system: sub-cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. Expression of SNAP-tag fusion proteins is described in the documentation supplied with SNAP-tag plasmids. The labeling of the fusion proteins with the SNAP-tag substrate is described below.
细胞通透性底物(SNAP-Cell）不仅能标记细胞内部也能标记细胞表面。激发光504nm，发射光532 nm。SNAP-Cell® 505-Star is a photostable green fluorescent substrate that can be used to label SNAP-tag® fusion proteins inside living cells, on cell surfaces, or in vitro. This cell-permeable substrate (CP-6-505) is based on the single isomer 6-carboxyrhodamine 110 and is suitable for standard fluorescein filter sets. It has an excitation maximum at 504 nm and an emission maximum at 532 nm. This package contains 50 nmol of SNAP-Cell 505-Star substrate, sufficient to make 10 ml of a 5 µM SNAP-tag fusion protein labeling solution.
The SNAP-tag protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is a protein based on human O6-alkylguanine-DNA alkyltransferase (hAGT). SNAP-tag substrates are fluorophores, biotin or beads conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag.
There are two steps to using this system: subcloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. Expression of SNAP-tag fusion proteins is described in the documentation supplied with SNAP-tag plasmids. The labeling of the fusion proteins with the SNAP-tag substrate is described below.
SNAP-Cell 647-SiR is a far-red fluorescent substrate that can be used to label SNAP-tag® fusion proteins inside living cells, on cell surfaces, or in vitro. This cell-permeable substrate (also termed SiR-SNAP)(1) is based on 6-carboxy-tetramethylsiliconrhodamine and is suitable for standard Cy5 filter sets. It has an excitation maximum at 645 nm and an emission maximum at 661 nm. This package contains 30 nmol of SNAP-Cell 647-SiR substrate, sufficient to make 10 ml of a 3 μM SNAP-tag fusion protein labeling solution.
The SNAP-tag protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is based on human O6-alkylguanine-DNA-alkyltransferase (hAGT). SNAP-tag substrates are fluorophores, biotin or beads conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag.
There are two steps to using this system: subcloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. Expression of SNAPtag fusion proteins is described in the documentation supplied with SNAP-tag plasmids. The labeling of the fusion proteins with the SNAP-tag substrate is described below.
细胞通透性底物(SNAP-Cell）不仅能标记细胞内部也能标记细胞表面。激发光500 nm，发射光532 nm。SNAP-Cell® Fluorescein is a green fluorescent substrate that can be used to label SNAP-tag® fusion proteins inside living cells or in vitro. This cell-permeable substrate (BG-Fluorescein) is based on diacetylfluorescein and is suitable for standard fluorescein filter sets. Diacetylfuorescein is essentially non-fluorescent, but it becomes fluorescent inside the cell when it is hydrolyzed by non-specific esterases, yielding fluorescein. It has an excitation maximum at 500 nm and an emission maximum at 532 nm. This substrate has limited photostability. If this presents a problem, we recommend using SNAP-Cell 505, which has similar spectral characteristics but much greater photostability. This package contains 50 nmol of SNAP-Cell Fluorescein substrate, sufficient to make 10 ml of a 5 µM SNAP-tag fusion protein labeling solution.
The SNAP-tag protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is a small protein based on mammalian O6-alkylguanine-DNA-alkyltransferase (AGT). SNAP-tag substrates are derivatives of benzylpurines and benzylchloropyrimidines, In the labeling reaction, the substituted benzyl group of the substrate becomes covalently attached to the SNAP-tag.
There are two steps to using this system: sub-cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. Expression of SNAP-tag fusion proteins is described in the documentation supplied with SNAP-tag plasmids. The labeling of fusion proteins with the SNAP-tag substrate is described below.
细胞通透性底物(SNAP-Cell）不仅能标记细胞内部也能标记细胞表面。激发光554nm，发射光580nm。SNAP-Cell® TMR-Star is a red fluorescent substrate that can be used to label SNAP-tag® fusion proteins inside living cells, on cell surfaces, or in vitro. This cell-permeable substrate (CP-6-TMR) is based on the single isomer 6-carboxytetramethylrhodamine and is suitable for standard rhodamine filter sets. It has an excitation maximum at 554 nm and an emission maximum at 580 nm. This package contains 30 nmol of SNAP-Cell TMR-Star substrate, sufficient to make 10 ml of a 3 µM SNAP-tag fusion protein labeling solution.
The SNAP-tag protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is a small protein based on mammalian O6-alkylguanine-DNA alkyltransferase (AGT). SNAP-tag substrates are derivatives of benzylpurines and benzylchloropyrimidines. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag.
There are two steps to using this system: subcloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. Expression of SNAP-tag fusion proteins is described in the documentation supplied with SNAP-tag plasmids. The labeling of the fusion proteins with the SNAP-tag substrate is described below.
细胞通透性底物(SNAP-Cell）不仅能标记细胞内部也能标记细胞表面。激发光490 nm，发射光514 nm。SNAP-Cell Oregon Green is a photostable green fluorescent substrate that can be used to label SNAP-tag® fusion proteins inside living cells. This cell-permeable substrate (BG-Oregon Green) is based on the Invitrogen dye, Oregon Green and is suitable for standard fluorescein filter sets. It has an excitation maximum at 490 nm and an emission maximum at 514 nm. Conjugates of Oregon Green are more photostable than those of fluorescein, and their fluorescence properties are essentially pH insensitive in the physiological pH range. This package contains 50 nmol of SNAP-Cell Oregon Green substrate, sufficient to make 10 ml of a 5 µM SNAP-tag fusion protein labeling solution.
The SNAP-tag is a novel tool for protein research, allowing the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is based on mammalian O6-alkylguanine-DNA-alkyltransferase (AGT). SNAP-tag substrates are derivatives of benzylguanines and benzylchloropyrimidines. In the labeling reaction, the dye-substituted benzyl group of the substrate becomes covalently attached to the SNAP-tag.
There are two steps to using this system: subcloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. Expression of SNAP-tag fusion proteins is described in the documentation supplied with SNAP-tag plasmids. The labeling of the fusion proteins with the SNAP-tag substrate is described below.
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