Capillary electrophoresis for the analysis of the effect of sample preparation on early stages of Aß1-40 aggregation.

Aggregation of the amyloid-ß protein (Aß) contributes to the neurodegeneration characteristic of Alzheimer's disease. Of particular importance are the early stages of aggregation, which involve the formation of soluble oligomers and protofibrils. In these studies, we demonstrate the potential for CE with UV detection using a polyethylene oxide separation matrix to identify the evolution of various oligomeric species of Aß1-40 . To demonstrate the efficacy of this technique, UV-CE was utilized to compare two methods commonly used to prepare Aß for aggregation experiments and their effect on the formation of early aggregates. SEC-purified Aß1-40 initially contained more small species, including monomer, than did freshly dissolved Aß1-40 pretreated with hexafluoroisopropanol. Strikingly, the lag time to oligomer formation for SEC-isolated Aß1-40 samples was ∼23 h shorter compared to freshly dissolved Aß1-40 samples. Furthermore, oligomers formed from the aggregation of SEC-purified Aß1-40 persisted within solution for a longer period of time. These results indicate that the initial sample preparation has a drastic influence on the early stages of Aß1-40 aggregation. This is the first report of the use of UV-CE with a separation matrix to study the effect of sample preparation on early aggregation of Aß1-40 . UV-CE was also used in parallel with dot blot analysis and inhibitory compounds to discern structural characteristics of individual oligomer peaks, demonstrating the capacity of UV-CE as a complimentary technique to further understand the aggregation process.

Unraveling the early events of amyloid-ß protein (Aß) aggregation: techniques for the determination of Aß aggregate size.

The aggregation of proteins into insoluble amyloid fibrils coincides with the onset of numerous diseases. An array of techniques is available to study the different stages of the amyloid aggregation process. Recently, emphasis has been placed upon the analysis of oligomeric amyloid species, which have been hypothesized to play a key role in disease progression. This paper reviews techniques utilized to study aggregation of the amyloid-ß protein (Aß) associated with Alzheimer's disease. In particular, the review focuses on techniques that provide information about the size or quantity of oligomeric Aß species formed during the early stages of aggregation, including native-PAGE, SDS-PAGE, Western blotting, capillary electrophoresis, mass spectrometry, fluorescence correlation spectroscopy, light scattering, size exclusion chromatography, centrifugation, enzyme-linked immunosorbent assay, and dot blotting.