Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils
Figure 2
Amyloid fibrils maintained their amyloid architecture after proteolytic digestion and acetone extraction.
(A) Aβ1–40, α-syn or gelsolin peptides (65 µg/ml) in a fibrillar (upper gel) or soluble (lower gel) state were incubated in the absence or presence of 0.13 µg/ml (1∶500, w/w) proteinase K (PK) for 2 h at 42°C. The digestion was conducted in 50 mM sodium phosphate, pH 7.4, 150 mM NaCl buffer. The reaction was stopped by boiling the samples in Laemmli buffer with 2% SDS and the samples were resolved by 16% SDS-PAGE. Western blot using 6E10 (Aβ1–40), syn-1 (α-syn) or a gelsolin-specific antibody is presented. (B) The same reaction described in panel A was performed in the presence of 20 µM of thioflavin T (ThT) and the florescence was monitored every 10 min. Ex = 440 nm and Em = 485 nm. (C) Aβ1–40 amyloid fibrils at 65 µg/ml concentration were diluted in 1 volume (1 V) of PBS, hexane, acetone or chloroform and centrifuged (16,000 g) for 10 min at 4°C. The pellet was resuspended in phosphate buffer with 20 µM ThT and the fluorescence measured. An aliquot of undiluted/uncentrifuged fibrils was used as the load. Ex = 450 nm and Em = 465–520 nm.