Assaying multiple biochemical variables from the same tissue sample
Assaying multiple biochemical variables from the same tissue sample
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DOI：
10.1016/j.jneumeth.2010.06.023
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发表时间：
2010-08-30
影响因子：
3
通讯作者：
Zigmond, Michael J.
中科院分区：
文献类型：
--
作者：
Leak, Rehana K.;Castro, Sandra L.;Zigmond, Michael J.
实验通常涉及多种分析，如神经递质和蛋白质的分析，这可能需要不同的初始样品制备。通常，这是通过使用不同的动物或来自同一动物的不同组织样本来完成的。这两种方法都使分析之间的比较更加多变，并大大增加了工作量和/或成本。利用从大鼠纹状体收集的组织和与帕金森病研究特别相关的分子，我们表明，在引用到适当的浓缩溶液（例如HClO4和裂解缓冲液）之前，在水中对组织进行超声处理，可以从相同的初始样品中进行几种类型的测量。多巴胺及其代谢物同质香草酸、血清素及其代谢物5-羟基吲哚乙酸、酪氨酸羟化酶及其Ser19和Ser31位点的磷酸化以及多巴胺转运体均未受影响。然而，phospho-Akt水平略有下降，phospho-ERK1/2有下降趋势。我们还提出了一种简单的技术，通过向动物心脏灌注磷酸酶抑制剂NaF来保持ERK1/2等蛋白的磷酸化状态。然而，多巴胺代谢物二羟苯乙酸（DOPAC）水平在两种技术下均升高。这里报告的一般原则可能适用于其他大脑区域，促进变量的多重比较，提高效率，降低成本。(C) 2010 Elsevier B.V.版权所有
Experiments often involve multiple analyses, such as assays of neurotransmitters and proteins, and this can require different initial sample preparations. Typically, this is accomplished by using different animals or different tissue samples from the same animal. Either approach renders comparisons between assays more variable and greatly increases the effort and/or cost. Using tissue collected from rat striatum and molecules of special relevance to studies of Parkinson's disease, we show that tissue sonication in water prior to aliquoting into the appropriate concentrated solutions (e.g. HClO4 and lysis buffers) permits several types of measurements to be made from the same initial samples. Dopamine and its metabolite homovanillic acid, serotonin and its metabolite 5-hydroxyindoleacetic acid, tyrosine hydroxylase and its phosphorylation at Ser19 and Ser31, and the dopamine transporter were unaffected. However, phospho-Akt levels fell slightly and phospho-ERK1/2 tended to drop. We also present a simple technique to preserve phosphorylation state of proteins such as ERK1/2 by perfusing animals through the heart with a phosphatase inhibitor, NaF. Dopamine metabolite dihydroxyphenyl acetic acid (DOPAC) levels were raised with both techniques, however. The general principles reported here are likely to apply to other brain regions, facilitate multiple comparisons of variables, increase efficiency, and decrease costs. (C) 2010 Elsevier B.V. All rights reserved.