天然腔肠素(Coelenterazine native),又称腔肠素游离酸,是海肾荧光素酶(Rluc)和Gaussia荧光素酶(Gluc)等多种荧光素酶的作用底物,也是水母发光蛋白的辅助因子,应用非常广泛,可用于检测活细胞中钙离子浓度,基因报告分析,BRET(生物发光共振能量转移)研究,ELISA,HTS,以及组织或细胞中ROS水平的化学发光检测。
英文别名(English synonym) | Renilla luciferin;Coelenterazine free base |
CAS号(CAS NO.) | 55779-48-1 |
分子式(Formula) | C26H21N3O3 |
分子量(Molecular weight) | 423.46g/mol |
外观(Appearance) | 黄色固体 |
溶解性(Solubility) | 溶于甲醇和乙醇,不溶于DMSO |
纯度(Purity)(TLC) | >97% |
1、使用腔肠素产品探究可激活的报告系统 Rluc-SMN的体内成像效果
图1 体内成像结果展示
冰袋运输;粉末-20℃避光干燥保存,最好保存在惰性气体环境下,避免接触空气;长期保存于-70℃。有效期2年。
Q:腔肠素作用原理?
A:腔肠素可作为许多荧光素酶的底物,比如海肾荧光素酶(Rluc),Gaussia 分泌型荧光素酶
(Gluc),以及包括水母发光蛋白(aequorin)和薮枝螅发光蛋白(Obelia)在内的光蛋白
(Photoproteins)。其发光原理是:以腔肠素为底物的荧光素酶在有分子氧的条件下,氧化腔肠素,产生高能量的中间产物,并在此过程中发射蓝色光,峰值发射波长约为 450~480 nm。
Q:腔肠素溶液如何配制?
A:腔肠素溶液配制方式来源文献,仅供参考,具体工作浓度建议参考文献做梯度摸索最佳浓度。母液:称取 1mg 腔肠素粉末直接溶解于 197µl 酸化的甲醇溶液( 100% 甲醇中含有20µl/ml 3M 或者 6M HCl),配制成 12 mM (~5mg/ml,5×) 的腔肠素储存液。 工作液:如体外:10 5 次方的细胞数可用 10 μ M 。
[1] Xu P, Huang S, Zhang H, et al. Structural insights into the lipid and ligand regulation of serotonin receptors. Nature. 2021;592(7854):469-473. doi:10.1038/s41586-021-03376-8(IF:49.962)
[2] Stauch B, Johansson LC, McCorvy JD, et al. Structural basis of ligand recognition at the human MT1 melatonin receptor [published correction appears in Nature. 2019 May 3;:]. Nature. 2019;569(7755):284-288. doi:10.1038/s41586-019-1141-3(IF:43.070)
[3] Xu P, Huang S, Mao C, et al. Structures of the human dopamine D3 receptor-Gi complexes. Mol Cell. 2021;81(6):1147-1159.e4. doi:10.1016/j.molcel.2021.01.003(IF:17.970)
[4] Tan Y, Xu P, Huang S, et al. Structural insights into the ligand binding and Gi coupling of serotonin receptor 5-HT5A. Cell Discov. 2022;8(1):50. Published 2022 May 24. doi:10.1038/s41421-022-00412-3(IF:10.849)
[5] Ji M, Wang X, Zheng H, et al. A Secreted Reporter for Blood Monitoring of Pyroptotic Cell Death [published correction appears in Anal Chem. 2021 Mar 2;93(8):4142]. Anal Chem. 2020;92(23):15565-15572. doi:10.1021/acs.analchem.0c03629(IF:6.785)
[6] Zheng H, Chen S, Wang X, Xie J, Tian J, Wang F. Intron Retained Bioluminescence Reporter for Real-Time Imaging of Pre-mRNA Splicing in Living Subjects. Anal Chem. 2019;91(19):12392-12398. doi:10.1021/acs.analchem.9b02935(IF:6.350)
[7] Guo B, Shi X, Ma Z, Ji M, Tang C, Wang F. A ratiometric dual luciferase reporter for quantitative monitoring of pre-mRNA splicing efficiency in vivo. J Biol Chem. 2021;297(2):100933. doi:10.1016/j.jbc.2021.100933(IF:5.157)
[8] Hu F, Zhang Y, Liu Q, Wang Z. PurA facilitates Edwardsiella piscicida to escape NF-κB signaling activation. Fish Shellfish Immunol. 2022;124:254-260. doi:10.1016/j.fsi.2022.04.001(IF:4.581)
[9] Xie J, Zheng H, Chen S, Shi X, Mao W, Wang F. Rational Design of an Activatable Reporter for Quantitative Imaging of RNA Aberrant Splicing In Vivo. Mol Ther Methods Clin Dev. 2020;17:904-911. Published 2020 Apr 18. doi:10.1016/j.omtm.2020.04.007(IF:4.533)
[10] Zheng H, Wang X, Chen S, et al. Real-Time Functional Bioimaging of Neuron-Specific MicroRNA Dynamics during Neuronal Differentiation Using a Dual Luciferase Reporter. ACS Chem Neurosci. 2019;10(3):1696-1705. doi:10.1021/acschemneuro.8b00614(IF:4.211)