Redefining Western Blot Standards: A Novel Approach for the Reliable Detection of Amyloid-Beta Oligomers (A𝜷Os) in Alzheimer’s Research

Neurotoxic amyloid-beta-oligomers (AβOs) accumulate in patients with Alzheimer’s Disease, driving cognitive decline and dementia. Determining their size via Western Blots (WB) could help create targeted therapies, yet exposure to detergent SDS could potentially lead to significant changes in the subunit association. Moreover, improper sample or antibody concentrations can cause primary antibody cross- linking, resulting in misleading signals (e.g., a stronger signal in wild-type controls that lack AβOs). Such inconsistencies have led researchers to overlook WB for AβO studies. This study adjusts SDS levels in the sample and running buffers and sample and antibody concentrations to minimize oligomer dissociation and antibody cross-linking, re-establishing WB’s viability for AβO research. Our preliminary results indicate that when using modified initial conditions - 0.0375% SDS Running Buffer, 0.0% SDS Sample Buffer, 3.75μg sample, and 0.1μg/ml NU2 antibody concentration - for the WB on mice samples, the expected results (low signal in wild-type samples, strong signal in 5xFAD) can be consistently obtained. This indicates that removing SDS from the sample buffer allows WB to be used on AβOs accurately while maintaining its high overall resolution. These findings can help optimize WB for AβO research and, generally, for studying molecules that self-associate.