Fig. 6.

Validation of enriched proteins using five-step sequential centrifugation.A, workflow of sequential extraction protocol used to create detergent-insoluble fractions from a pooled AD brain sample. B, silver-stained SDS-PAGE gel of the total homogenate (input) and different insoluble fractions obtained from sequential centrifugation steps (∼1 μg per sample). C, schematic diagram showing the profiling of AD brain fractions. A total of six biological samples were used, including the total homogenate (input) and five insoluble fractions, each with three replicates. D, clustering analysis of the proteins identified with three replicates. E and F, examples of DEP distribution in the six samples. G, Western blot validation of Aβ enrichment in P1 and P5 in different samples (∼5 μg protein per sample except ∼10 μg of the input). Ponceau S staining of the blot indicated the loading level. Aβ, amyloid-beta; AD, Alzheimer's disease; DEP, differentially expressed protein.