Reagents, ELISA and y-secretase Assay Core

试剂、ELISA 和 γ-分泌酶检测核心

• 批准号: 7920125
• 负责人: Weiming Xia
• 金额: $ 17.74万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7920125
• 关键词: Address; Alzheimer' s Disease; Animals; Antibodies; Area; Binding; Biological Assay; Biology; Brain; C-terminal; Cataloging; Catalogs; Cell Line; Cells; Collaborations; Collection; Communities; Complementary DNA; Complex; Conditioned Culture Media; Country; Cultured Cells; Development; Embryo; 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; Pathway interactions; Peptide Hydrolases; Peptides; Plasma; Plasmids; Play; Productivity; Proteins; Reagent; Regulation; Research; Research Personnel; Resources; Role; Scientist; Senior Scientist; Series; Services; Source; Specificity; Subcellular Fractions; Testing; Transgenic Mice; Transgenic Organisms; Work; cross reactivity; design; dimer; genetic regulatory protein; in vitro activity; mouse model; mutant; notch protein; novel; novel strategies; presenilin; 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的主要作用包括通过ELISA定量AS水平，开发新的抗AS抗体，以及通过ELISA检测AS水平。 ELISA和γ-分泌酶测定，以及该计划广泛试剂的维护和分发。 首先，核心B将继续对单体总AS、A61-40、ASx-40、ASx-42和AS 1 - 40进行AS ELISA。 42为所有参与该计划的研究人员。我们将测量单体AS在 来自稳定细胞系和原代神经元培养物的条件培养基，细胞裂解物，亚细胞级分， 小鼠和人脑匀浆以及转基因小鼠血浆。核心将提供有效的 以及获得所有四个项目和动物的AS定量测量的方便手段 核心其次，核心B将开发用于测量可溶性寡聚AS物质的特异性ELISA。低 分子量AS寡聚体在神经元代谢中起重要作用， 二聚体/三聚体/四聚体将通过我们新开发的ELISA来测量。第三，核心B将建立新的 定量Notch和APLP 2的γ-分泌酶切割的ELISA方法。AS样肽的水平将 通过ELISA通过定量嵌合APP-Notch和APP-APLP 2的γ-分泌酶切割来测量 proteins.第四，核心B将进行标准的γ-分泌酶活性测定，并开发新的方法， 调节体外γ-分泌酶活性。此外，核心B将在以下条件下进行体外γ-分泌酶测定： 项目研究者提出的新条件。最后，核心B将继续负责 维持和提供广泛的共享细胞系、DMA构建体和该计划的抗体， 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充当重要的中心 储存了大量非常有用的试剂，这些试剂是在过去几年中产生和表征的。 过去二十年来，参与该计划项目的调查人员。这些试剂包括数百种 cDNA构建体，几十个稳定转染的哺乳动物细胞系和大量的 特异性多克隆和单克隆抗体。核心B的使命是极大地促进 合作和整体实验效率在计划项目。