Figure 1.
Preparation of different variants of tau fibrils. (A) Sedimentation test for passages 1–6 of repetitively self-seeded T40 fibrillization without heparin. Supernatant (S) and pellet (P) fractions were resolved on SDS-PAGE and stained by Coomassie blue. Data are representative of more than three independent fibrillization series. (B) Sedimentation test for T40 fibrils induced with and without heparin; three different preparations are shown for each category. The heparin-free fibrils were from passages 9 or 10 of repetitively self-seeded fibrillization as shown in A. (C) Negative staining EM images for T40 fibrils induced with and without heparin after sonication. Bar, 100 nm. (D) A schematic diagram summarizing the main steps of tau PHF purification from AD brains. (E and F) Different fractions from PHF purification (refer to the schematic in D) were immunoblotted with 17025 (a polyclonal pan-tau antibody) and PHF-1 (a monoclonal antibody specific for tau phosphorylated at S396/S404). (F) Ponceau S staining for the final purification steps revealed further removal of contaminants from the sarkosyl pellet. The final supernatant (fraction 3, red in D) is the fraction used in the study and referred to as AD-tau. Data are representative of at least eight independent extractions (Table S2). (G) Silver staining of AD-tau shows prominent bands between 50 and 75 kD, recognized by a panel of tau antibodies, including T14 (a monoclonal antibody specific for human tau), 17025, PHF-1, RD3 (a 3R tau-specific monoclonal antibody), and an anti-4R tau polyclonal antibody. Representative preparations from the frontal cortices of three AD cases are shown (1a and 1b are two different preparations from the same case). (H) Negative staining EM images for AD-tau with (+) and without (−) sonication. Bar, 100 nm. (A, B, and E–G) Molecular mass is indicated in kilodaltons.