SDS-PAGE/immunoblot detection of Abeta multimers in human cortical tissue homogenates using antigen-epitope retrieval.
The anomalous folding and polymerization of the beta-amyloid (Abeta) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis(1). Abeta is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into beta-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 Abeta multimers are more toxic than fibrillar Abeta amyloid(2). The identification and quantification of low- and high-molecular weight multimeric Abeta 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 proteopathies(3). Naturally occurring Abeta multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with Abeta-specific antibodies. However, the separation and detection of multimeric Abeta 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 Abeta peptide. Using this protocol, we consistently detect Abeta monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.
Rosen
Rebecca F
RF
Yerkes National Primate Research Center, Emory University.
Tomidokoro
Yasushi
Y
Ghiso
Jorge A
JA
Walker
Lary C
LC
D000544:Alzheimer Disease / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000032:analysis* / Q000276:immunology; D015153:Blotting, Western / Q000379:methods*; D002540:Cerebral Cortex / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D000939:Epitopes / Q000032:analysis / Q000276:immunology; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis* / Q000276:immunology
D016229:Amyloid beta-Peptides; D000939:Epitopes; D010446:Peptide Fragments; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42)