FlAsH-EDT2
(Synonyms: 荧光含砷螺旋粘结剂/卢米奥绿色,Fluorescein Arsenical Helix Binder, Lumio Green) 目录号 : GC43674A fluorescent probe that tags proteins in cells
Cas No.:212118-77-9
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
FlAsH-EDT2 is a pro-fluorescent, membrane-permeable biarsenical compound that binds covalently to a tetracysteine sequence (CCPGCC), which is engineered into target proteins.[1],[2] It binds proteins that have the CCPGCC tag almost immediately after translation.[2] FlAsH-EDT2 is commonly used to study protein trafficking, folding, and interactions in living cells or cell lysates.[2],[3],[4] This green-emitting fluorophore is excited at 508 nm, with emission at 528 nm.[4]
Reference:
[1]. Griffin, B.A., Adams, S.R., and Tsien, R.Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 287(5374), 269-272 (1998).
[2]. Rudner, L., Nydegger, S., Coren.L.V., et al. Dynamic fluorescent imaging of human immunodeficiency virus type 1 gag in live cells by biarsenical labeling. J.Virol. 79(7), 4055-4065 (2005).
[3]. Luedtke, N.W., Dexter, R.J., Fried, D.B., et al. Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH. Nat.Chem.Biol. 3(12), 779-784 (2007).
[4]. Perdios, L., Bunney.T.D., Warren, S.C., et al. Time-resolved FRET reports FGFR1 dimerization and formation of a complex with its effector PLCγ1. Adv.Biol.Regul. 60, 6-13 (2016).
| Cas No. | 212118-77-9 | SDF | |
| 别名 | 荧光含砷螺旋粘结剂/卢米奥绿色,Fluorescein Arsenical Helix Binder, Lumio Green | ||
| 化学名 | 4',5'-bis(1,3,2-dithiarsolan-2-yl)-3',6'-dihydroxy-spiro[isobenzofuran-1(3H),9'-[9H]xanthen]-3-one | ||
| Canonical SMILES | OC1=C([As]2SCCS2)C3=C(C4(C(C=CC=C5)=C5C(O4)=O)C6=CC=C(O)C([As]7SCCS7)=C6O3)C=C1 | ||
| 分子式 | C24H18As2O5S4 | 分子量 | 664.5 |
| 溶解度 | DMF: 15 mg/ml,DMSO: 20 mg/ml,DMSO:PBS (pH 7.2) (1:10): 0.09 mg/ml | 储存条件 | Store at -20°C,protect from light |
| 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 | 1.5049 mL | 7.5245 mL | 15.0489 mL |
| 5 mM | 0.301 mL | 1.5049 mL | 3.0098 mL |
| 10 mM | 0.1505 mL | 0.7524 mL | 1.5049 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 protein-labeling reagent FlAsH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins
Pflugers Arch 2001 Sep;442(6):859-66.PMID:11680618DOI:10.1007/s004240100619.
FLASH-EDT2--4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)2--has been reported to fluoresce only after binding with high affinity to a specific tetracysteine motif (CCXXCC, "Cys4") and thus to provide a technique for labeling recombinant proteins in vivo (Griffin et al. Science 281:269-272). We have attempted to use FlAsH-EDT2 as a site-specific label of the II-III loop of the dihydropyridine receptor (DHPR) in skeletal muscle. Upon expression in dysgenic myotubes (which lack endogenous alpha1s), an alpha1s mutated to contain CCRECC in the II-III loop was able to produce L-type calcium currents and to mediate skeletal-type excitation-contraction (EC) coupling, but FlAsH-EDT2 labeling revealed no difference from non-transfected dysgenic myotubes. HeLa-S3 cells transfected with Cys4-containing calmodulin were significantly more fluorescent than non-transfected cells, whereas the difference between transfected and non-transfected cells was less apparent for CHO-K and HEK 293 cells. Because the fluorescence of non-transfected cells increased substantially after treatment with FlAsH-EDT2, it suggested the possibility that FLASH binds to endogenous cysteine-containing proteins. This finding was confirmed in cuvette experiments in which FlAsH-EDT2 fluorescence was observed after FLASH-EDT, was added to protein homogenates from myotubes or cell lines. The enhanced fluorescence was abolished by pretreatment of cells or cell homogenates with coumarine maleimide (CPM), which modifies cysteine residues covalently. Thus, enhanced FLASH fluorescence appears to occur both after binding to an introduced Cys4 motif and to endogenous, cysteine-containing proteins. Therefore, FlAsH-EDT2 may be useful only for labeling those recombinant proteins that express at a very high level.
Direct quantification of cytosolic delivery of drug nanocarriers using FlAsH-EDT2
Nanomedicine 2023 Jan;47:102626.PMID:36356708DOI:10.1016/j.nano.2022.102626.
The delivery of therapeutics across the cell membrane and into the cytoplasm is a major challenge that limits the development of new therapies. This challenge is compounded by the lack of a general assay for cytosolic delivery. Here we develop this assay based on the pro-fluorophore CrAsH-EDT2, and provide cytosolic penetration results for a variety of drug delivery agents (polyethyleneimine, poly-arginine, Ferritin, poly [maleic anhydride-alt-isobutene] grafted with dodecylamine, and cationic liposomes) into HeLa and T98G cells. Our results show that this method can be widely applicable to different cells and drug delivery agents, and yield statistically robust results. We later use this method to optimize and improve a model drug delivery agent's (Ferritin) cytosolic penetration.
Preparation of the membrane-permeant biarsenicals FlAsH-EDT2 and ReAsH-EDT2 for fluorescent labeling of tetracysteine-tagged proteins
Nat Protoc 2008;3(9):1527-34.PMID:18772880DOI:10.1038/nprot.2008.144.
The membrane-permeant fluorogenic biarsenicals FlAsH-EDT(2) and ReAsH-EDT(2) can be prepared in good yields by a straightforward two-step procedure from the inexpensive precursor dyes fluorescein and resorufin, respectively. Handling of toxic reagents such as arsenic trichloride is minimized so the synthesis can be carried out in a typical chemistry laboratory, usually taking about 2-3 d. A wide range of other biarsenical reagents and intermediates that also bind to tetracysteine-tagged (CysCysProGlyCysCys) proteins can be prepared similarly using this general procedure.
Therapeutic and analytical applications of arsenic binding to proteins
Metallomics 2015 Jan;7(1):39-55.PMID:25356501DOI:10.1039/c4mt00222a.
Arsenic binding to proteins plays a pivotal role in the health effects of arsenic. Further knowledge of arsenic binding to proteins will advance the development of bioanalytical techniques and therapeutic drugs. This review summarizes recent work on arsenic-based drugs, imaging of cellular events, capture and purification of arsenic-binding proteins, and biosensing of arsenic. Binding of arsenic to the promyelocytic leukemia fusion oncoprotein (PML-RARα) is a plausible mode of action leading to the successful treatment of acute promyelocytic leukemia (APL). Identification of other oncoproteins critical to other cancers and the development of various arsenicals and targeted delivery systems are promising approaches to the treatment of other types of cancers. Techniques for capture, purification, and identification of arsenic-binding proteins make use of specific binding between trivalent arsenicals and the thiols in proteins. Biarsenical probes, such as FlAsH-EDT2 and ReAsH-EDT2, coupled with tetracysteine tags that are genetically incorporated into the target proteins, are used for site-specific fluorescence labelling and imaging of the target proteins in living cells. These allow protein dynamics and protein-protein interactions to be studied. Arsenic affinity chromatography is useful for purification of thiol-containing proteins, and its combination with mass spectrometry provides a targeted proteomic approach for studying the interactions between arsenicals and proteins in cells. Arsenic biosensors evolved from the knowledge of arsenic resistance and arsenic binding to proteins in bacteria, and have now been developed into analytical techniques that are suitable for the detection of arsenic in the field. Examples in the four areas, arsenic-based drugs, imaging of cellular events, purification of specific proteins, and arsenic biosensors, demonstrate important therapeutic and analytical applications of arsenic protein binding.
Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH
Nat Chem Biol 2007 Dec;3(12):779-84.PMID:17982447DOI:10.1038/nchembio.2007.49.
Recombinant polypeptides and protein domains containing two cysteine pairs located distal in primary sequence but proximal in the native folded or assembled state are labeled selectively in vitro and in mammalian cells using the profluorescent biarsenical reagents FlAsH-EDT2 and ReAsH-EDT2. This strategy, termed bipartite tetracysteine display, enables the detection of protein-protein interactions and alternative protein conformations in live cells. As proof of principle, we show that the equilibrium stability and fluorescence intensity of polypeptide-biarsenical complexes correlates with the thermodynamic stability of the protein fold or assembly. Destabilized protein variants form less stable and less bright biarsenical complexes, which allows discrimination of live cells expressing folded polypeptide and protein domains from those containing disruptive point mutations. Bipartite tetracysteine display may provide a means to detect early protein misfolding events associated with Alzheimer's disease, Parkinson's disease and cystic fibrosis; it may also enable high-throughput screening of compounds that stabilize discrete protein folds.