Coelenteramine 400a hydrochloride  (Synonyms: Coelenterazine 400a hydrochloride; Bisdeoxycoelenterazine hydrochloride)

Coelenteramine (Coelenterazine) 400a hydrochloride, a derivative of Coelenterazine, is a Renilla luciferase (RLuc) substrate. In the presence of Coelenteramine 400a hydrochloride, RLuc can emit blue light at 395 nm^[1][2]. Coelenteramine 400a hydrochloride will causes color change in the bioluminescence reaction of Rluc by replacing the sulfur and oxygen heteroatoms of the methylene bridge. Coelenteramine 400a hydrochloride provides higher signal resolution and can be used in the research of bioluminescence resonance energy transfer (BRET)^[3].

发光反应^[4]
1.溶液制备：
1) 准备 10 μL 浓度为 1.0-5.0 mM 的 Coelenteramine 400a 溶液。
2) 准备 10 μL 浓度为 2.01 mg/mL 的 Renilla luciferase (RLuc8.6-53) 溶液。
3) 将上述两种溶液与 200 μL 缓冲液混合。
2. 具体染色步骤：
1) 使用配备液氮冷却 CCD 探测器的多通道光谱仪记录混合溶液的生物发光光谱。光谱仪的绝对光谱灵敏度需事先用 500 W 光谱辐照度标准灯校准。
2) 采用定制的配备光电倍增管 (PMT，H11890 - 01，Hamamatsu Photonics，Japan) 的光度计测定生物发光反应的相关参数。光度计的绝对响应度需用积分球光谱仪校准，通过光度计测量的相对计数值与积分球测量的绝对值绘制线性图来确定。
3) 在试管中，将 5-15 μL 浓度为 10 nM (总计 50-150 fmol) 的 Coelenteramine 400a 溶液预先放入光度计中，然后注入 150 μL 浓度为 20 μg/mL 的荧光素酶溶液 (在 0.1 M GTA 缓冲液中，pH 分别为 6.0、7.0 和 8.0) 启动反应。监测反应直至发光反应完全，从获得的总光子数和荧光素分子数计算生物发光反应量子产率。
4) 使用 Lineweaver-Burk 图计算底物 Coelenteramine 400a 的米氏常数 (K_m)。计算催化常数 (k_cat)。使用 Quantaurus-QY 在激发光设置为 300 nm 的条件下测量相应氧化产物 Coelenteramide 400a (CTMD 400a) 的荧光光谱和绝对荧光量子产率，测量时 CTMD 400a 溶液在 DMSO 中的浓度为 20 μM。

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

427.93

C₂₆H₂₂ClN₃O

2320429-05-6

Room temperature in continental US; may vary elsewhere.

Please store the product under the recommended conditions in the Certificate of Analysis.

• [1]. Levi J, et, al. Bisdeoxycoelenterazine derivatives for improvement of bioluminescence resonance energy transfer assays. J Am Chem Soc. 2007 Oct 3;129(39):11900-1.  [Content Brief]

  [2]. Bertrand L, et, al. The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRS). J Recept Signal Transduct Res. Feb-Nov 2002;22(1-4):533-41.  [Content Brief]

  [3]. Yuan M L, et al. Luminescence of coelenterazine derivatives with C-8 extended electronic conjugation[J]. Chinese Chemical Letters, 2016, 27(4): 550-554.

  [4]. Ikeda Y, et al. Quantitative evaluation of luminescence intensity from enzymatic luminescence reaction of coelenterazine and analogues. Journal of Photochemistry and Photobiology A: Chemistry, 2020, 394: 112459.