Cell-based assays for regulators of tau biology.
Understanding the molecular mechanisms of Alzheimer's disease (AD) is a challenging endeavor, namely, due to the fact that the disease only occurs in the central nervous system of elderly humans. Thus, model systems simply do not accurately portray this cellular landscape. While we cannot ask many mechanistic questions using the human brain as our test subject, cell culture techniques that have emerged at least provide us with the ability to pose fundamental biochemical questions, which may ultimately lead to translational outcomes for AD. In particular, the intracellular microtubule-associated protein tau that accumulates in AD is found in normal cells. Manipulating these cells may allow us to address basic questions about tau biology that would provide novel therapeutic strategies down the road. Here, we describe several techniques to explore tau cell biology using the Odyssey(®) Infrared Imaging system (Odyssey system) from LI-COR Biosciences. We provide a detailed protocol on how to perform a scalable drug screening assay called the In-Cell Western and follow-up these screens with standard Western analysis to confirm whether "hits" are valid by more traditional means. We provide some tips on where mistakes are most likely to occur, and we interpret our standard Western data, providing some estimation as to the composition of the banding pattern that is typical for this enigmatic protein.
Jinwal
Umesh K
UK
Department of Molecular Medicine, Byrd Alzheimer's Institute, University of South Florida, Tampa, FL, USA.
Dickey
Chad A
CA
D001681:Biological Assay / Q000379:methods*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D006801:Humans; D016875:tau Proteins / Q000378:metabolism*
D016875:tau Proteins