5-Ph-IAA
目录号 : GC61525
5-Ph-IAA 是 IAA 的衍生物。 5-Ph-IAA 是一种配体，与 OsTIR1 (F74G) 突变体一起建立了生长素诱导型 degron 2 (AID2) 系统。 AID2 诱导 mAID 融合蛋白的快速有效消耗，以研究活细胞中的蛋白质功能，从而抑制肿瘤。
Cas No.:168649-23-8
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
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| Cell experiment [1]: | |
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Cell lines |
HCT116 cell |
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Preparation Method |
HCT116 cells were seeded at 1 × 105 cells/well in a six-well plate and grown for 2 days. Cells were treated with 0.5 μg/mL of doxycycline for 24 h, and then 1 μM 5-Ph-IAA was added. |
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Reaction Conditions |
1μM 5-Ph-IAA |
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Applications |
The mAID-fused targets were rapidly degraded by the addition of 1μM 5-Ph-IAA. |
| Animal experiment [2]: | |
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Animal models |
Balb/c-nu female(7 weeks old) weighing 16-20 g |
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Preparation Method |
Indicated HCT116 lines (1 × 10 5 cells for mAID-BRD4 and 2 × 10 5 cells for TOP2A-mAC) were resuspended in 0.1 ml of HBSS containing 0.05 ml of Matrigel. The suspension was injected into the both sides of flank. Six or 7 days after 5-Ph-IAA injection, the mice were randomized and treated daily with the indicated dose of 5-Ph-IAA by IP injection for additional 6 or 7 days. Tumour volume was measured on the indicated days. At the end of the experiment, xenograft tumour was removed and weighed. |
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Dosage form |
5-Ph-IAA by IP injection，0, 1, 3, 10 mg/kg |
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Applications |
significant tumour suppression of mAID-BRD4 xenografts at all doses of 5-Ph-IAA treatment |
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References: [1]. Yesbolatova A, Saito Y, Kitamoto N, Makino-Itou H, Ajima R, Nakano R, Nakaoka H, Fukui K, Gamo K, Tominari Y, Takeuchi H, Saga Y, Hayashi KI, Kanemaki MT. The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice. Nat Commun. 2020 Nov 11;11(1):5701. |
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5-phenyl-indole-3-acetic acid (5-Ph-IAA) is a derivative of IAA. 5-Ph-IAA is a ligand to establish the auxin-induced degron 2 system (AID2) together with the OsTIR1 (F74G) mutant. Using AID2, degron fusion proteins can now be more precisely controlled, enabling target protein degradation with a half-life of 10 to 45 minutes by adding low doses of 5-Ph-IAA [1]. 5-Ph-IAA shows higher affinity to AtTIR1(F79G).
The mAID-fused targets were rapidly degraded by the addition of 1 μM 5-Ph-IAA and showed significant tumour suppression of mAID-BRD4 xenografts at all doses of 5-Ph-IAA treatment including 0, 1, 3, 10 mg/kg [1]. The treatment with 50 mM 5-Ph-IAA caused a rapid decrease in fluorescence intensity in many embryos (78%, 18/23 embryos), but 5 mM 5-Ph-IAA did not. 5-Ph-IAA can be used for loss-of-function experiments in C. elegans embryos, although treatment with 5-Ph-IAA is not always effective, possibly because the C. elegans embryo develops within the eggshell, which blocks its permeability to many compounds [2].
References:
[1].Yesbolatova A, Saito Y, Kitamoto N, Makino-Itou H, Ajima R, Nakano R, Nakaoka H, Fukui K, Gamo K, Tominari Y, Takeuchi H, Saga Y, Hayashi KI, Kanemaki MT. The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice. Nat Commun. 2020 Nov 11;11(1):5701.
[2].Negishi T, Kitagawa S, Horii N, Tanaka Y, Haruta N, Sugimoto A, Sawa H, Hayashi KI, Harata M, Kanemaki MT. The auxin-inducible degron 2 (AID2) system enables controlled protein knockdown during embryogenesis and development in Caenorhabditis elegans. Genetics. 2022 Feb 4;220(2):iyab218.
5-phenyl-indole-3-acetic acid (5-Ph-IAA) 是 IAA 的衍生物。 5-Ph-IAA 是与 OsTIR1 (F74G) 突变体一起建立生长素诱导的 degron 2 系统 (AID2) 的配体。使用 AID2，现在可以更精确地控制 degron 融合蛋白，通过添加低剂量的 5-Ph-IAA [1] 使目标蛋白降解的半衰期为 10 至 45 分钟。 5-Ph-IAA对AtTIR1(F79G)具有更高的亲和力。
通过添加 1 μM 5-Ph-IAA，mAID 融合靶标会迅速降解，并且在所有剂量的 5-Ph-IAA 处理（包括 0、1、3、10毫克/千克 [1]。用 50 mM 5-Ph-IAA 处理导致许多胚胎（78%，18/23 胚胎）的荧光强度快速降低，但 5 mM 5-Ph-IAA 没有。 5-Ph-IAA 可用于秀丽隐杆线虫胚胎的功能丧失实验，尽管用 5-Ph-IAA 处理并不总是有效，这可能是因为秀丽隐杆线虫胚胎在蛋壳内发育，阻碍了其渗透性许多化合物 [2].
| Cas No. | 168649-23-8 | SDF | |
| Canonical SMILES | O=C(O)CC1=CNC2=C1C=C(C3=CC=CC=C3)C=C2 | ||
| 分子式 | C16H13NO2 | 分子量 | 251.28 |
| 溶解度 | DMSO: 125 mg/mL (497.45 mM) | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 3.9796 mL | 19.8981 mL | 39.7962 mL |
| 5 mM | 0.7959 mL | 3.9796 mL | 7.9592 mL |
| 10 mM | 0.398 mL | 1.9898 mL | 3.9796 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。
The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice
Nat Commun 2020 Nov 11;11(1):5701.PMID:33177522DOI:10.1038/s41467-020-19532-z.
Protein knockdown using the auxin-inducible degron (AID) technology is useful to study protein function in living cells because it induces rapid depletion, which makes it possible to observe an immediate phenotype. However, the current AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin. These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and to apply this method to mice. Here, we overcome these problems by taking advantage of a bump-and-hole approach to establish the AID version 2 (AID2) system. AID2, which employs an OsTIR1(F74G) mutant and a ligand, 5-Ph-IAA, shows no detectable leaky degradation, requires a 670-times lower ligand concentration, and achieves even quicker degradation than the conventional AID. We demonstrate successful generation of human cell mutants for genes that were previously difficult to deal with, and show that AID2 achieves rapid target depletion not only in yeast and mammalian cells, but also in mice.
Targeted Protein Depletion Using the Auxin-Inducible Degron 2 (AID2) System
Curr Protoc 2021 Aug;1(8):e219.PMID:34370399DOI:10.1002/cpz1.219.
Targeted protein depletion using a conditional degron is a powerful method to probe the role of proteins in living cells because of the speed with which depletion can be induced and its reversibility. The auxin-inducible degron (AID) is one of the most common degron-based technologies used in cell biology. We recently established an improved system, called AID2, which involves expressing a mutant E3 ligase subunit, OsTIR1(F74G), and fusing a protein of interest to the mini-AID (mAID) tag, and that employs a new and more potent ligand, 5-phenyl-indole-3-acetic acid (5-Ph-IAA). The AID2 system overcomes some of the drawbacks associated with the original AID system, i.e., leaky degradation without auxin and the requirement of high auxin doses. With AID2 it is, therefore, now possible to control a degron-fused protein more precisely, enabling target proteins to be degraded with a half-life of 10 to 45 min via the addition of a low dose of 5-Ph-IAA. Importantly, in AID2, it is not necessary to control the expression of OsTIR1(F74G) for suppressing leaky degradation and a parental cell line constitutively expressing OsTIR1(F74G) can be used for the generation of multiple mAID-tagged proteins. Here, we describe a protocol for the tagging of endogenous proteins with mAID in diploid HCT116 cells. Our protocol can be applied to other mammalian cell lines and will enhance the utility of AID2 for studying protein functions in living cells. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Generation of a parental HCT116 cell line expressing OsTIR1(F74G) Basic Protocol 2: Construction of CRISPR and donor plasmids for tagging endogenous genes Basic Protocol 3: Generation of cell lines expressing a protein of interest fused with mAID.
The auxin-inducible degron 2 (AID2) system enables controlled protein knockdown during embryogenesis and development in Caenorhabditis elegans
Genetics 2022 Feb 4;220(2):iyab218.PMID:34865044DOI:10.1093/genetics/iyab218.
Targeted protein degradation using the auxin-inducible degron (AID) system is garnering attention in the research field of Caenorhabditis elegans, because of the rapid and efficient target depletion it affords, which can be controlled by treating the animals with the phytohormone auxin. However, the current AID system has drawbacks, i.e., leaky degradation in the absence of auxin and the requirement for high auxin doses. Furthermore, it is challenging to deplete degron-fused proteins in embryos because of their eggshell, which blocks auxin permeability. Here, we apply an improved AID2 system utilizing AtTIR1(F79G) and 5-phenyl-indole-3-acetic acid (5-Ph-IAA) to C. elegans and demonstrated that it confers better degradation control vs the previous system by suppressing leaky degradation and inducing sharp degradation using 1,300-fold lower 5-Ph-IAA doses. We successfully degraded the endogenous histone H2A.Z protein fused to an mAID degron and disclosed its requirement in larval growth and reproduction, regardless of the presence of maternally inherited H2A.Z molecules. Moreover, we developed an eggshell-permeable 5-Ph-IAA analog, 5-Ph-IAA-AM, that affords an enhanced degradation in laid embryos. Our improved system will contribute to the disclosure of the roles of proteins in C. elegans, in particular those that are involved in embryogenesis and development, through temporally controlled protein degradation.
An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in Caenorhabditis elegans
Genetics 2022 Feb 4;220(2):iyab174.PMID:34739048DOI:10.1093/genetics/iyab174.
The auxin-inducible degradation system in C. elegans allows for spatial and temporal control of protein degradation via heterologous expression of a single Arabidopsis thaliana F-box protein, transport inhibitor response 1 (AtTIR1). In this system, exogenous auxin (Indole-3-acetic acid; IAA) enhances the ability of AtTIR1 to function as a substrate recognition component that adapts engineered degron-tagged proteins to the endogenous C. elegans E3 ubiquitin ligases complex [SKR-1/2-CUL-1-F-box (SCF)], targeting them for degradation by the proteosome. While this system has been employed to dissect the developmental functions of many C. elegans proteins, we have found that several auxin-inducible degron (AID)-tagged proteins are constitutively degraded by AtTIR1 in the absence of auxin, leading to undesired loss-of-function phenotypes. In this manuscript, we adapt an orthogonal auxin derivative/mutant AtTIR1 pair [C. elegans AID version 2 (C.e.AIDv2)] that transforms the specificity of allosteric regulation of TIR1 from IAA to one that is dependent on an auxin derivative harboring a bulky aryl group (5-Ph-IAA). We find that a mutant AtTIR1(F79G) allele that alters the ligand-binding interface of TIR1 dramatically reduces ligand-independent degradation of multiple AID*-tagged proteins. In addition to solving the ectopic degradation problem for some AID-targets, the addition of 5-Ph-IAA to culture media of animals expressing AtTIR1(F79G) leads to more penetrant loss-of-function phenotypes for AID*-tagged proteins than those elicited by the AtTIR1-IAA pairing at similar auxin analog concentrations. The improved specificity and efficacy afforded by the mutant AtTIR1(F79G) allele expand the utility of the AID system and broaden the number of proteins that can be effectively targeted with it.