Determination of intracellular concentrations of the TRPM2 agonist ADP-ribose by reversed-phase HPLC
Determination of intracellular concentrations of the TRPM2 agonist ADP-ribose by reversed-phase HPLC
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DOI：
10.1016/j.jchromb.2005.05.002
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发表时间：
2005-07-25
影响因子：
3
通讯作者：
Guse, AH
中科院分区：
文献类型：
--
作者：
Gasser, A;Guse, AH
由于 NAD 代谢物 ADP-核糖 (ADPR) 最近作为假定的信使而受到关注，因此建立了一种通过反相 HPLC 定量细胞内 ADPR 的方法。用三氯乙酸提取细胞核苷酸，并使用强阴离子交换基质通过固相萃取纯化粗细胞提取物。优化提取程序后，使用两种不同的反相柱（C18 与 C12）以离子对模式操作测定细胞 ADPR 水平。人Jurkat T淋巴细胞和鼠BW5147胸腺细胞中的细胞内ADPR浓度分别测定为44+/-11μM和73+/-11μM。 (c) 2005 Elsevier B.V. 保留所有权利。
Since the NAD metabolite ADP-ribose (ADPR) has recently gained attention as a putative messenger, a method was established for the quantification of intracellular ADPR by reversed-phase HPLC. Cellular nucleotides were extracted with trichloroacetic acid, and crude cell extracts purified by solid phase extraction using a strong anion exchange matrix. After optimization of the extraction procedure, cellular ADPR levels were determined using two different reversed-phase columns (C18 versus C12), operated in ion pair mode. Intracellular ADPR concentrations in human Jurkat T-lymphocytes and murine BW5147 thymocytes were determined to be 44 +/- 11 mu M and 73 +/- 11 mu M, respectively. (c) 2005 Elsevier B.V. All rights reserved.