Protein extraction, Aβ fractionation

The tissue was thawed, weighed and processed by sonication in Triton-X-lysis buffer containing 1% Triton^TM X-100, 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 2 mM EDTA, 1 mM DTT, phosphatase inhibitor cocktails 2 and 3 (Sigma) and complete^TM Mini EDTA-free Protease Inhibitor Cocktail tablet (Roche) at a ratio of 1:10 (1 mg tissue = 10 μL Triton-X-lysis buffer). The resulting total lysates were assayed for protein concentration using the Pierce^TM BCA protein assay. Triton-X-soluble, SDS-soluble and urea-soluble fractions were generated for cortical and hippocampal tissue. Equal amounts of Triton-X-lysate protein (850 µg) in a final volume of 100 µL were centrifuged at 100,000 × g for 60 min at 4 °C. The supernatant was collected as the Triton-X-soluble fraction. Pellets were washed with Triton-X-lysis buffer and then re-suspended in equals volumes of Triton-X-lysis buffer containing 1% SDS and centrifuged at 100,000 × g for 60 min at 4 °C to generate the SDS-soluble fraction. The remaining pellets were washed with Triton-X-lysis buffer containing 1% SDS and dissolved in buffer containing 30 mM Tris-HCl, pH 8.5, 7 M urea, 2 M thiourea and 4% CHAPS and centrifuged at 100,000 × g for 60 min at 4 °C to generate the urea-soluble fraction.