Biochemical Core

本项目资助的生物化学核心实验室将提供高度整合的 专业的、装备精良的专业单位，能够执行项目调查人员要求的各种放射免疫分析、生化和分子程序。由R01支持的任何一个调查员实验室都不能配备和配备这一资源。核心将执行所有的分析技术：1)用放射化学高效液相色谱法测定细胞色素P450和一氧化氮合酶活性。2)用LC/MS测定组织样品、脑脊液和微透析液中的20-HETE和EETs；3)用LC？MS/MS鉴定由细胞色素P4F和细胞色素P4X酶产生的新代谢物。此外，我们还将用免疫印迹和免疫组织化学方法检测细胞色素P450和一氧化氮合酶蛋白。实时RT-PCR和原位杂交的分子测量将如项目中概述的那样进行。 获得中央设施的分析专门知识和设备对每个拟议的项目都是至关重要的，成功完成拟议的研究所需的大量生化和分子测定也证明了这一点。

The Biochemical Core Laboratory of this Program Project Grant will provide a consolidated highly specialized well-equipped and professional unit capable of performing a variety of radioimmunoassay, biochemical and molecular procedures as required by project investigators. No single investigator laboratory supported by R01 would be capable of equipping and staffing this resource. The Core will carry out all the analytical techniques: 1) Assay of CYP and NOS activities by radiochemical HPLC. 2) Measurement of 20- HETE and EETs in tissue samples, CSF and microdialysates by LC/MS. 3) Identification of novel metabolites generated by CYP4F and CYP4X enzymes by LC?MS/MS. This Core is also equipped to measure IP3, DAG, microglial and astroglial products in the brain and cerebral vasculature. In addition we will measure CYP and NOS protein using western blots and immunohistochemistry. Molecular measurements with real time RT-PCR and in situ hybridizations will be carried out as outlined in the projects. Access to the analytical expertise and equipment in the central facility is essential to each of the proposed projects and justified by the large number of biochemical and molecular determinations required for successful completion of the proposed studies.