3X FLAG Peptide
目录号 : GP101493X FLAG Peptide 是一种合成肽，具有 3 次重复的 DYKXXD 基序。
Sample solution is provided at 25 µL, 10mM.
| ELISA experiment [1]: | |
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Preparation method |
The solubility of this peptide in sterile water is >10 mM. Stock solution should be splited and stored at -80°C for several months. |
| Applications |
3-Flag peptide has found widespread use as a mild purification reagent for Flag-epitope tagged recombinant proteins. Although its affinity columns release monovalent flagged proteins in the absence of calcium, the antibody retains substantial affinity for the Flag sequence even in metal-free conditions, so that it has been impossible to use it to develop a metal-sensitive ELISA assay. This is due to the ability of the antibody to remain bound to polyvalent surface-coated antigen, for instance, when Flagged proteins are bound to ELISA plates or blotting filters. The resultant antigen polyvalence raises the avidity of the Flag antibody to a point where the reaction is essentially calcium-independent. However, when the antibody itself was made monovalent, by proteolytic cleavage to the Fab, this situation was reversed and the ELISA reaction became calcium-dependent. This new metal-dependent ELISA assay was used to explore the metal requirements of the antibody in detail. Among divalent metals, binding tapered off with increasing radius above that of calcium, or with decreasing radius below that of calcium. Several smaller metals, such as nickel, acted as inhibitors of the binding reaction. Substantial binding was demonstrated for heavy metals such as cadmium, lanthanum and samarium. Because it is of interest to use this antibody for the co-crystallization of recombinant Flag-fusion proteins, the ability to bind heavy metals was a significant finding. |
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References: 1. Hopp TP1, Gallis B, Prickett KS. Metal-binding properties of a calcium-dependent monoclonal antibody. Mol Immunol. 1996 May-Jun;33(7-8):601-8. |
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The FLAG-tag system utilizes a short, hydrophilic 8- amino-acid peptide that is fused to the protein of interest1. The FLAG peptide binds to the antibody M1. Whether binding is calcium-dependent manner2 or –independent3 remains controversial. A disadvantage of the system is that the monoclonalantibody purification matrix is not as stable as others. In general, small tags can be detected with specific monoclonal antibodies.
To improve the detection of the FLAG tag the 3x FLAG system has been developed. This threetandem FLAG epitope is hydrophilic, 22-amino-acids long and can detect up to 10 fmol of expressed fusion protein. The FLAG-tagged maltodextrin-binding protein of Pyrococcus furiosus has been crystallized4 and the quality of the crystals was very similar to that of crystals of untagged protein.
Finally, the FLAG-tag can be removed by treatment with enterokinase, which is specific for the five C-terminal amino acids of the peptide sequence5.
FLAG标签系统(FLAG-tag system)利用一个短的、亲水性的8个氨基酸多肽与感兴趣的蛋白质融合 [1]。FLAG 肽与抗体 M1 结合。结合是钙依赖性方式 [2] 还是非依赖性方式 [3] 仍然存在争议。该系统的缺点是单克隆抗体纯化基质不像其他基质那样稳定。一般来说，小标签可以用特异性单克隆抗体检测。
为了提高FLAG标签的检测，3x FLAG系统被开发。这种三联 FLAG 表位是亲水性的，长 22 个氨基酸，可以检测高达 10 fmol 的表达融合蛋白。Pyrococcus furiosus 的 FLAG 标记的麦芽糖糊精结合蛋白已经结晶，晶体的质量与未标记蛋白质的晶体非常相似[4]。
最后，FLAG标签可以通过酶切割除，该酶专门切割多肽序列的五个羧基末端氨基酸 [5]。
References:
1. Hopp TP, Prickett KS, Price VL, Libby RT, March CJ, Ceretti DP, Urdal DL, Conlon PJ (1988) A short polypeptide marker sequence useful for recombinant protein identification and purification. Bio/Technology 6:1204–1210.
2. Hopp TP, Gallis B, Prikett KS (1996) Metal-binding properties of a calcium dependent monoclonal antibody. Mol Immunol 33:601–608.
3. Einhauer A, Jungbauer A (2000) Kinetics and thermodynamical properties of the monoclonal antibody M1 directed against the FLAG peptide. 20th International symposium on the separation of proteins, peptides, and polynucleotides (ISPPP). Lublijana, Slovenia, November 5–8, 2000.
4. Bucher MH, Evdokimov AG, Waugh DS (2002) Differential effects of short affinity tags on the crystallization of Pyrococcus furiosus maltodextrin-binding protein. Biol Cryst 58:392–397.
5. Maroux S, Baratti J, Desnuelle P (1971) Purification and specificity of procine enterokinase. J Biol Chem 246:5031–5039.
| Cas No. | 402750-12-3 | SDF | |
| 别名 | H-Met-Asp-Tyr-Lys-Asp-His-Asp-Gly-Asp-Tyr-Lys-Asp-His-Asp-Ile-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-OH | ||
| 化学名 | 3X FLAG Peptide | ||
| Canonical SMILES | CCC(C)C(C(=O)NC(CC(=O)O)C(=O)NC(CC1=CC=C(C=C1)O)C(=O)NC(CCCCN)C(=O)NC(CC(=O)O)C(=O)NC(CC(=O)O)C(=O)NC(CC(=O)O)C(=O)NC(CC(=O)O)C(=O)NC(CCCCN)C(=O)O)NC(=O)C(CC(=O)O)NC(=O)C(CC2=CNC=N2)NC(=O)C(CC(=O)O)NC(=O)C(CCCCN)NC(=O)C(CC3=CC=C(C=C3)O)NC(=O)C(CC(=O)O)NC( | ||
| 分子式 | C120H169N31O49S | 分子量 | 2861.87 |
| 溶解度 | ≥ 143.1mg/mL in DMSO, <14.35mg/mL in EtOH, ≥ 143.4mg/mL in Water with gentle warming | 储存条件 | Desiccate at -20°C |
| General tips | For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while. | ||
| Shipping Condition | Evaluation sample solution : ship with blue ice All other available size: ship with RT , or blue ice upon request |
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| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 0.3494 mL | 1.7471 mL | 3.4942 mL |
| 5 mM | 0.0699 mL | 0.3494 mL | 0.6988 mL |
| 10 mM | 0.0349 mL | 0.1747 mL | 0.3494 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。