Purification of recombinant tau E14

The recombinant 6xHis-tau E14 was expressed and purified from Sf9 insect cells using the Baculovirus expression system as previously described^16,84. Briefly, the cells from two 500 ml cultures were combined, pelleted, and lysed with Purification Lysis Buffer (Lysis Buffer plus 1 mM Benzamidine, 1 mM PMSF, 14 mM 2-mercaptoethanol) by vigorous pipetting, followed by snap-freezing in liquid nitrogen. The frozen lysate was quickly thawed, sonicated using a Sonifier cell disruptor B-30 (Branson Sonic Power) and boiled for 10 min at 95 °C. The lysates were centrifuged (100,000g, 1 h, 4 °C) and the supernatant was loaded on a Ni²⁺ affinity column (HisTrap FF, GE Healthcare Life Sciences), The purified protein was concentrated using 10 kDa cutoff Amicon Ultra-4 concentrators and loaded on a Superdex 200 10/300 GL (GE Healthcare Life Sciences) size exclusion chromatography column with HK Buffer (25 mM HEPES–KOH pH 7.4, 100 mM KCl, freshly supplemented with 1 mM DTT) as buffer exchange. The fractions were analysed for purity and quality through SDS-PAGE and total protein staining with Coomassie InstantBlue. The affinity purification and size exclusion chromatography steps were performed using the ÄKTA system.