Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils
Figure 3
Effect of proteinase K digestion and acetone precipitation on the protein content of a complex biological extract.
(A and B) The complex biological extract was obtained by mechanical disruption of wild type C. elegans worms followed by a brief centrifugation (700 g for 3 min) to remove unlysed worms. Aβ1–40 fibrils (0.2% w/w protein concentration) were added to the worm post debris supernatant (PDS) and the samples were digested with PK (1∶500) for 2 h at 42°C followed by acetone precipitation. An aliquot before PK digestion (load), after PK digestion (+ PK) and after PK digestion and acetone precipitation (+ PK/acetone) were resolved by SDS-PAGE (A) or the protein was quantified by BCA assay (B). In the panel A, the upper gel is silver stained and the lower gel is a Western blot for Aβ using the 6E10 antibody. (C–F) TEM images of PDS. PDS was incubated in the absence (C) or in the presence of 0.2% Aβ1–40 fibrils (E) before the PK/acetone step. PDS incubated in the absence (D) or in the presence of 0.2% Aβ1–40 fibrils (F) was digested with PK and precipitated with acetone. Note that amyloid fibrils are present only in the samples to which Aβ1–40 fibrils were added (E and F).