Furimazine (PBI 3939)

Furimazine是一种咪唑并吡嗪酮类的发光底物。在哺乳动物细胞中，NanoLuc(Nluc)与Furimazine联用，产生更亮的发光，比带Coelenterazine的Oluc-19高250万倍。

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1mg

￥2900.00

2320.00

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5mg

￥4125.00

3300.00

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10mg

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25mg

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50mg

￥16537.00

13230.00

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Background

Furimazine is an imidazopyrazinone substrate. NanoLuc (Nluc) paired with Furimazine produced 2.5 million-fold brighter luminescence in mammalian cells relative to Oluc-19 with Coelenterazine.

Furimazine is a Coelenterazine analogue.The apparent Km for purified NanoLuc (Nluc) using either Furimazine or Coelenterazine is ~10 μM, while the maximum luminescence (i.e., apparent Vmax) is ~30-fold higher for Furimazine than for native Coelenterazine. Measurement of luminescence intensity from cells expressing Nluc reveals that maximal signal is attained at about 10-20 μM Furimazine[1].

[1]. Hall MP, et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol. 2012 Nov 16;7(11):1848-57.

Protocol

Kinase experiment:

Random libraries are generated by error-prone PCR (average of 2-3 mutations per clone). Library 1 (phase 1; template=Oluc-N166R) is screened (4,400 variants) with Coelenterazine. Library 2 (phase 2; template=C1A4E) is screened (4,400 variants) with 11 novel Coelenterazine analogues: 3840, 3841, 3842, 3857, 3880, 3881, 3886, 3887, 3889, 3897, and 3900. The 11 analogues represent substitutions at positions 2, 6, and 8 and are considered to be representative of the entire set of 24 compounds; 2,200 variants are screened with compounds 3896 and 3894. All hits (improved luminescence) are screened again with the remaining Coelenterazine analogues. Library 3 (phase 3; template=C1A4E+Q18L/K33N/F54I/F68Y/L72Q/M75K/I90V) was screened in the context of a mouse Id-X-HaloTag (where X=library) using Coelenterazine and Furimazine. Library screens are performed on a Freedom robotic workstation as follows: induced bacterial cultures (in 96-well microtiter plates) are lysed with a buffer containing 300 mM HEPES pH 8, 200 mM thiourea, 0.3X Passive Lysis Buffer, 0.3 mg/mL lysozyme, and 0.002 units of RQ1 DNase. Assay reagent containing 1 mM CDTA, 150 mM KCl, 10 mM DTT, 0.5% (v/v) Tergitol, and 20 μM substrate is then added to equal volumes of lysate. Samples are measured on a GENios Pro luminometer[1].

参考文献:

[1]. Hall MP, et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol. 2012 Nov 16;7(11):1848-57.