Affiliations: [a] Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA | [b] Litwin-Zucker Center for Research in Alzheimer's Disease, Feinstein Institute for Medical Research, North Shore/LIJ Health System, Manhasset, NY, USA
Correspondence:
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Correspondence to: Peter Davies, Litwin-Zucker Center for Research in Alzheimer's Disease, Feinstein Institute for Medical Research, North Shore/LIJ Health System, Manhasset, NY 11030, USA. Tel.: +1 516 562 3416; E-mail: pdavies@nshs.edu.
Abstract: In Alzheimer's disease (AD) and tauopathies, tau becomes hyperphosphorylated, undergoes a conformational change, and becomes aggregated and insoluble. There are three methods commonly used to study the insoluble tau fraction, two that utilize detergents (Sarkosyl and RIPA) and another that does not (insoluble). However, these methods require large amounts of homogenate for a relatively low yield of the insoluble fraction, which can be problematic when dealing with small tissue samples. Furthermore, the most common way of analyzing this material is through densitometry of immunoblots, offering only semiquantitative measurements. We provide a comparison of the three methods commonly used (Sarksoyl, RIPA, and insoluble) through immunoblot and ELISA analyses. Finally, we tested a new method to determine aggregated tau levels, utilizing a monoantibody tau ELISA. The insoluble fractions of four different mouse models (P301 L, htau, wild type, and knockout) as well as human AD and control brains were examined. There were significant correlations between the three insoluble methods for both total tau and pS396/404 tau measured by immunoblot or ELISA analyses. Additionally, the results from the ELISA method correlated significantly with those from immunoblot analyses. Finally, the monoantibody assay on the lysate significantly correlated with the total tau ELISAs performed on the three insoluble preparations. Taken together, these results suggest that all three insoluble preparation methods offer similar results for measuring insoluble tau in either mouse or human brains. In addition the new monoantibody ELISA offers a simple quantitative method to measure the amount of aggregated tau in both human and mouse brains.
Keywords: Alzheimer's disease, ELISA, immunoblotting, methodology, tau protein, transgenic mice
