An efficient extraction method for quantitation of adenosine triphosphate in mammalian tissues and cells
An efficient extraction method for quantitation of adenosine triphosphate in mammalian tissues and cells
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DOI：
10.1016/j.aca.2012.03.022
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发表时间：
2012-05-21
影响因子：
6.2
通讯作者：
Kido, Hiroshi
中科院分区：
文献类型：
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作者：
Chida, Junji;Yamane, Kazuhiko;Kido, Hiroshi
萤火虫生物发光广泛应用于生物材料中 5'-三磷酸腺苷 (ATP) 水平的测量。对于组织和细胞中的此类检测，必须在初始步骤中从蛋白质中提取 ATP，并且提取效率是检测准确性的主要决定因素。市售 ATP 检测试剂盒中推荐的提取试剂是离液试剂、三氯乙酸 (TCA)、高氯酸 (PCA) 和乙二醇 (EG)，它们通过蛋白质沉淀和/或核苷酸酶失活来提取核苷酸。我们发现这些试剂对于测量蛋白质浓度相对较低的材料（例如血细胞、培养细胞和细菌）中的 ATP 水平特别有用。然而，这些方法不适用于从高蛋白浓度的组织中提取ATP，因为一些ATP可能在均质和提取过程中与不溶的蛋白共沉淀，并且也可能通过酸提取物中和而沉淀。在这里，我们发现基于苯酚的提取方法显着增加了从组织中提取的 ATP 和其他核苷酸。此外，在荧光素-荧光素酶测定步骤之前，苯酚提取不需要中和。通过荧光素酶测定分析，Tris-EDTA 饱和苯酚（苯酚-TE）提取的各种组织中的 ATP 水平比 TCA 提取的高 17.8 倍以上，比 EG 提取物高 550 倍以上。在这里，我们报告了一种简单、快速、可靠的苯酚-TE 提取程序，用于通过荧光素酶测定测量组织和细胞中的 ATP。 (C) 2012 Elsevier B.V. 保留所有权利。
Firefly bioluminescence is widely used in the measurement of adenosine 5'-triphosphate (ATP) levels in biological materials. For such assays in tissues and cells, ATP must be extracted away from protein in the initial step and extraction efficacy is the main determinant of the assay accuracy. Extraction reagents recommended in the commercially available ATP assay kits are chaotropic reagents, trichloroacetic acid (TCA), perchloric acid (PCA), and ethylene glycol (EG), which extract nucleotides through protein precipitation and/or nucleotidase inactivation. We found that these reagents are particularly useful for measuring ATP levels in materials with relatively low protein concentrations such as blood cells, cultured cells, and bacteria. However, these methods are not suitable for ATP extraction from tissues with high protein concentrations, because some ATP may be co-precipitated with the insolubilized protein during homogenization and extraction, and it could also be precipitated by neutralization in the acid extracts. Here we found that a phenol-based extraction method markedly increased the ATP and other nucleotides extracted from tissues. In addition, phenol extraction does not require neutralization before the luciferin-luciferase assay step. ATP levels analyzed by luciferase assay in various tissues extracted by Tris-EDTA-saturated phenol (phenol-TE) were over 17.8-fold higher than those extracted by TCA and over 550-fold higher than those in EG extracts. Here we report a simple, rapid, and reliable phenol-TE extraction procedure for ATP measurement in tissues and cells by luciferase assay. (C) 2012 Elsevier B.V. All rights reserved.