TRFS-green
TRFS-green 是一种高选择性关断荧光探针，用于对活细胞中的硫氧还蛋白还原酶 (TrxR) 进行成像。
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包装价格促销价数量
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1mg(10 mM * 240 in DMSO)￥900.00550.00- +
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5mg￥2012.001100.00- +
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10mg￥3412.001900.00- +
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25mg￥7162.004000.00- +
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50mg￥12100.007500.00- +
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1g￥30000.000.00- +
- 货号: ajcx11636
- CAS: 1513848-14-0
- 别名:
- 分子式: C20H20N2O4S2
- 分子量: 416.51
- 纯度: ＞98%
- 溶解度: Soluble in DMSO
- 储存: Store at -20°C
- 库存: 现货
Background
The probe features a 1,2-dithiolane scaffold with a quenched naphthalimide fluorophore. TRFS-green displays a green fluorescence off-on change induced by the TrxR-mediated disulfide cleavage and subsequent intramolecular cyclization to liberate the masked naphthalimide fluorophore.
It was demonstrated in vitro that TRFS-green manifests high selectivity toward TrxR over other related enzymes and various small molecule thiols as well as biological reducing molecules. HPLC analyses indicated that TRFS-green was exclusively converted to naphthalimide catalyzed by TrxR. The ability in triggering on the fluorescence signal by cellular protein extracts correlates well with the endogenous TrxR activity in different cells. Furthermore, inhibition of TrxR by 2,4-dinitrochlorobenzene or depletion of TrxR by immunoprecipitation remarkably decreases the reduction of TRFS-green by cellular protein extracts. Finally, TRFS-green was successfully applied in imaging TrxR activity in living cells. The fluorescence signal of TRFS-green in living cells was inhibited by pretreating the cells with TrxR inhibitor in a dose-dependent manner, potentiating the development of living cell-based screening assay for identifying TrxR inhibitors.
体外研究：TRFS-green 显示出由 TrxR 介导的二硫键裂解和随后的分子内环化诱导的绿色荧光开关变化，以释放掩蔽的萘酰亚胺荧光团[1]。
当在 377 nm 处激发时，TRFS-green 在 ~480 nm 处会有微弱信号。用 TrxR 抑制剂以剂量依赖性方式预处理活细胞，可抑制活细胞中 TRFS-green 的荧光信号，从而促进基于活细胞的 TrxR 抑制剂筛选试验的发展[1]。
TRFS-green 对 GSH 或 Cys (细胞中的主要硫醇) 或内源性还原剂 (例如维生素 C 和 NADPH) 没有反应[1]。
测量粗细胞提取物中的 TrxR 活性[1]：将细胞裂解物与 TRFS-green 孵育适当的时间 (通常 1-2 小时)，并通过荧光计测定荧光强度 (Em=540 nm，Ex=440 nm)。还可以在给定的时间段内监测荧光的时间依赖性增加 (Em=540 nm，Ex=440 nm)。
TRFS-green (10 μM；2-4 h) 处理细胞后，可通过显微镜的绿色荧光通道观察[1]。
注意：为了获得清晰的荧光图像，建议在拍照前立即用 PBS 或新鲜培养基清洗细胞以去除残留的 TRFS-green。
TRFS-green 是 TrxR 的第一个具有绿色发射光的荧光探针。然而，TRFS-green 对酶的响应速度较慢，即使使用 三(2-羧乙基)膦(TCEP) 作为还原剂，也需要 2 小时以上才能达到最大荧光信号[2]。
[1]. Liangwei Zhang, et al. Highly selective off-on fluorescent probe for imaging thioredoxin reductase in living cells. J Am Chem Soc. 2014 Jan 8;136(1):226-33.
[2]. Xinming Li, et al. A fast and specific fluorescent probe for thioredoxin reductase that works via disulphide bond cleavage. Nat Commun. 2019 Jun 21;10(1):2745.
