Rita Costa, Frederico Ferreira-da-Silva, Maria J. Saraiva, and Isabel Cardos. Transthyretin Protects against A-Beta Peptide Toxicity by Proteolytic Cleavage of the Peptide: A Mechanism Sensitive to the Kunitz Protease Inhibitor.  PLoS ONE. 2008; 3(8): e2899. Published online 2008 August 6. doi: 10.1371/journal.pone.0002899.

...Human, recombinant or sera, TTR (15 µg) and A-Beta peptide (1–42) (2 µg) were incubated in 20 µl 50 mM Tris, pH 7.5, at 37°C for different periods of time, and then reactions were applied onto a 15% SDS-PAGE gel and visualized by coomassie blue staining. Alternatively, TTR incubated with A-Beta (1–42) and A-Beta alone, were run on a SDS-PAGE gel, transferred to nitrocellulose membranes, and A-Beta immunodetected using an anti-A-Beta antibody (BAM-10, Sigma). In another set of experiments, A-Beta was incubated for 6 hours at 37°C for aggregation purposes and then subsequently incubated with TTR. Results were visualized by immunoblot, as described above.

To evaluate possible inhibitors of A-Beta proteolysis by TTR, recombinant TTR (15 µg) was pre-incubated for 30 minutes at 37°C, either with 1 mM pefabloc, 0.33 µM αAPP of peptide encompassing aminoacids 18–688, containing the KPI domain (Neuromics) or with 0.33 µM αAPP peptide derived from the APP isoform 695 (without the KPI domain) (Sigma), both formed by a α-secretase cleavage. Then, A-Beta was added (2 µg) in a final volume of 20 µl, and further incubated for 6 hours at 37°C. Results were observed by immunoblot as described. Experiments were repeated at least twice...