SDS-PAGE/Immunoblot Detection of Aβ Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

• Type of Material: Article
• Authors: Rebecca F. Rosen, Emory University; Yasushi Tomidokoro, Tsukuba University; Jorge A. Ghiso, New York University; Lary C Walker, Emory University
• Language: English
• Date: 2010-04-23
• Publisher: Journal of Visualized Experiments (JoVE)
• Publication Version: Final Published Version
• Copyright Statement: © 2010, Journal of Visualized Experiments
• Final Published Version (URL): http://dx.doi.org/10.3791/1916
• Title of Journal or Parent Work: Journal of Visualized Experiments
• ISSN: 1940-087X
• Issue: 38
• Grant/Funding Information: Funding was provided by RR-00165, PO1AG026423, P50AG025688, AG030539, the Woodruff Foundation and the Emory University Research Committee.
• Research Categories: Biology, Neuroscience; Health Sciences, Pathology

Abstract

The anomalous folding and polymerization of the β-amyloid (Aβ) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis1. Aβ is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into β-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble Aβ multimers are more toxic than fibrillar Aβ amyloid2. The identification and quantification of low- and high-molecular weight multimeric Aβ species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies3. Naturally occurring Aβ multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with Aβ-specific antibodies. However, the separation and detection of multimeric Aβ requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the Aβ peptide. Using this protocol, we consistently detect Aβ monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.