Reagents, ELISA and y-secretase Assay Core

试剂、ELISA 和 γ-分泌酶检测核心

• 批准号: 7468591
• 负责人: Weiming Xia
• 金额: $ 17.22万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7468591
• 关键词: Address; Animals; Antibodies; Area; Binding; Biological Assay; Biology; Brain; C-terminal; Cataloging; Catalogs; Cell Line; Cells; Collaborations; Collection; Communities; Complementary DNA; Complex; Condition; Conditioned Culture Media; Country; Cultured Cells; Development; Embryo; Endopeptidases; Ensure; Environment; Enzyme-Linked Immunosorbent Assay; Epitopes; Equipment and supply inventories; Goals; Human; In Vitro; International; Laboratories; Maintenance; Mammalian Cell; Measurement; Measures; Metabolism; Methods; Mission; Molecular Weight; Monoclonal Antibodies; Mus; N-terminal; Neurodegenerative Disorders; Neurons; Numbers; Pathway interactions; Peptide Hydrolases; Peptides; Plasma; Plasmids; Play; Productivity; Proteins; Reagent; Regulation; Research; Research Personnel; Resources; Role; Scientist; Senior Scientist; Series; Services; Source; Specificity; Standards of Weights and Measures; Subcellular Fractions; Testing; Transgenic Mice; Transgenic Organisms; Work; cross reactivity; design; desire; dimer; genetic regulatory protein; mouse model; mutant; notch protein; novel; novel strategies; programs; repository; secretase; small molecule; stable cell line

The primary roles of Core B include the quantification of AS levels by ELISAs, the development of novel ELISA and y-secretase assays, and the maintenance and distribution of the Program's extensive reagents. First, Core B will continue to perform AS ELISAs for monomeric total AS, A61-40, ASx-40, ASx-42 and AS1- 42 for all investigators involved in the Program. We will measure the concentration of monomeric AS in conditioned media from stable cell lines and primary neuronal cultures, cell lysates, subcellular fractions, mouse and human brain homogenates, and transgenic mouse plasma. The Core will provide an effective and convenient means for obtaining quantitative measurements of AS for all four projects and the animal core. Second, Core B will develop a specific ELISA for measuring soluble oligomeric AS species. Low molecular weight AS oligomers play significant roles in neuronal metabolism, and the levels of soluble AS dimers/trimers/tetramers will be measured by our newly developed ELISA. Third, Core B will establish new ELISA methods to quantify the y-secretase cleavage of Notch and APLP2. Levels of AS-like peptides will be measured by ELISA by quantifying the y-secretase cleavages of chimeric APP-Notch and APP-APLP2 proteins. Fourth, Core B will perform standard y-secretase activity assay and develop new approaches to modulate the y-secretase activity in vitro. Further, Core B will carry out the in vitro y-secretase assay under new conditions proposed by a Program Investigator. Finally, Core B will continue to be responsible for maintaining and supplying the extensive shared cell lines, DMA constructs and antibodies of the Program to all of our investigators as well as to outside investigators, as in the past. Core B thus serves as a vital central repository for a large number of highly useful reagents that have been generated and characterized over the past two decades by the Investigators involved in this Program Project. These reagents include hundreds of cDNA constructs, several dozen stably-transfected mammalian cell lines and an extensive collection of specific polyclonal and monoclonal antibodies. The mission of Core B is to greatly facilitate ongoing collaborations and overall experimental efficiency within the Program Project.

核心B的主要作用包括用ELISA量化砷水平，开发新的 酶联免疫吸附试验和γ-分泌酶分析，以及该计划广泛试剂的维护和分发。 首先，核心B将继续作为单体总AS、A61-40、ASX-40、ASX-42和AS1-的ELISA发挥作用。 42适用于参与该方案的所有调查人员。我们将测量单体的浓度，如 来自稳定细胞系和原代神经元培养的条件培养液，细胞裂解物，亚细胞组分， 小鼠和人脑匀浆，以及转基因小鼠血浆。核心将提供一个有效的 以及方便地获得所有四个项目和动物的砷的定量测量的手段 核心。第二，Core B将开发一种专门的酶联免疫吸附试验，用于测量可溶低聚物的种类。低 作为寡聚体的分子量在神经元代谢中起着重要作用，而可溶性AS水平 二聚体/三聚体/四聚体将通过我们新开发的酶联免疫吸附试验进行检测。第三，核心B将建立新的 用ELISA法测定Notch和APLP2的γ-分泌酶活性。AS样多肽的水平将是 定量检测嵌合APP-Notch和APP-APLP2的γ-分泌酶裂解 蛋白质。第四，核心B将进行标准的γ-分泌酶活性分析，并开发新的方法来 体外调节γ-分泌酶活性。此外，Core B将在以下条件下进行体外γ-分泌酶测试 由项目调查员提出的新条件。最后，核心B将继续负责 维护和供应程序的广泛共享细胞系、DMA结构和抗体 我们所有的调查人员以及外部调查人员，就像过去一样。因此，核心B充当至关重要的中央 大量高度有用的试剂的存储库，这些试剂已在 参与该计划项目的研究人员在过去二十年中所做的工作。这些试剂包括数百种 Cdna构建，数十个稳定转染的哺乳动物细胞系和广泛的 特异性多克隆和单克隆抗体。核心B的使命是大大促进正在进行的 计划项目内的协作和总体实验效率。