Reagents, ELISA and y-secretase Assay Core

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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.