Biochemical Core

该计划的生物化学核心实验室项目资助将提供一个综合的高度 这是一个装备精良的专业化单位，能够按照项目调查人员的要求进行各种放射免疫测定、生物化学和分子程序。没有一个由R 01支持的研究者实验室能够配备和配备这一资源。核心将执行所有分析技术：1）通过放射化学HPLC测定γ-氨基丁酸和NOS活性。2)LC/MS法测定组织样品、CSF和微透析液中的20- HETE和E2。3）LC法鉴定CYP 4F和CYP 4X酶产生的新代谢产物？MS/MS。该核心还配备测量脑和脑血管系统中的IP 3、DAG、小胶质细胞和星形胶质细胞产物。此外，我们将使用蛋白质印迹和免疫组织化学来测量NOS和NOS蛋白。将按照项目所述，采用真实的实时RT-PCR和原位杂交进行分子测量。 在中心设施中获得分析专门知识和设备对每个拟议项目都是必不可少的，成功完成拟议研究所需的大量生物化学和分子测定证明了这一点。

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.