Quantifying Tau Protein Aggregation

Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was performed as previously described^{9 (link),26 (link)}. Briefly, brain extract normalized to contain 340 ng total tau was loaded onto a 1.5% agarose gel with Laemmli buffer (20 mmol l⁻¹ Tris-base, 200 mmol l⁻¹ glycine and 0.02% sodium dodecyl sulfate). The gel was run on ice at 35 V for 16 h and then transferred onto a polyvinylidene difluoride (PVDF) membrane overnight in TBS using filter paper and capillary action. The membrane was blocked in TBS containing 0.25% Tween 20 and 5% non-fat dry milk and incubated overnight with rabbit polyclonal anti-tau antibody (1:1,000; Dako). Horseradish-peroxidase-conjugated goat anti-rabbit IgG secondary antibody was applied in blocking solution on day 2 and was detected using chemiluminescent horseradish-peroxidase substrate (Thermo Fisher Scientific) and film (GE Healthcare Life Sciences). For quantification, grayscale images were scanned and imported into Fiji/ImageJ. Using the gel quantification densitometry tool, the gel was separated into 14 bands (bins) of equal size that were all quantified and plotted.