Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils
Figure 6
Immunoprecipitation of amyloid fibrils from tissue extracts of a C. elegans strain that overexpresses human Aβ1–42 peptide.
(A) Worm post debris supernatant (PDS) from N2 (wild type) or CL2006 (Aβ) worms were applied to SDS-PAGE (Load) or processed as described in Figure 5A (Eluate) before being applied to SDS-PAGE. N2 (wild type) worms were used at day 1 of adulthood, whereas CL2006 (Aβ) worms were used at days 1, 5 or 8 of adulthood. The gel was transferred to nitrocellulose membrane that was probed for Aβ (≈4 kDa) using the 6E10 antibody or for tubulin (≈55 kDa) as a loading control. The amount of sample applied to the gel was 10 fold higher for the eluate (10X) when compared with the load (1X). In lane 9, synthetic Aβ1–40 peptide (2 ng) was used as a standard for Aβ. Note that the peptide Aβ1–40 runs faster than the Aβ synthesized in the CL2006 worms. (B) Quantification of Aβ bands of panel (A). Since the eluate fractions do not contain tubulin, we normalized the eluate bands using the tubulin bands of the load samples. The quantification was made using Fiji software and the bars represent the standard deviation of two experiments.