TITLE: Biochemical detection of aggregated Tau
AUTHORS: Itika Saha, F. Ulrich Hartl, Mark S. Hipp

[DESCRIPTION]
This protocol describes the detection of aggregated Tau from HEK293 cells stably expressing and propagating aggregates of Tau repeat domain fused to YFP (Sanders et al. Neuron, 2014; Saha et al, BioRxiv, 2022). The filter trap assay is a modification of the membrane filter assay described previously (Wanker et al., Methods in Enzymology, 1999). 

[STEPS]
SECTION: Preparation of cell lysates
1. The protocol can be performed with freshly harvested cells or with frozen cell pellets. In either case, harvest cells by trypsinization and wash 1x with PBS before pelleting in 1.5 mL Eppendorf tubes.
SECTION: Preparation of cell lysates
2. Thaw frozen cell pellets on ice for at least 10 min.
SECTION: Preparation of cell lysates
3. For cell pellets harvested from 1 well of a 12-well plate, add 75 µL RIPA lysis buffer (Thermo) or 1% Triton X-100/PBS supplemented with protease inhibitor cocktail (Roche) and DNase, and resuspend pellets by pipetting.
SECTION: Preparation of cell lysates
4. When using 1% Triton X-100/PBS, briefly sonicate samples to lyse nuclei and achieve a homogenous lysate.
SECTION: Preparation of cell lysates
5. Incubate on ice for 30 min to 1 h.
SECTION: Preparation of cell lysates
6. Centrifuge lysates at 1,000 x g for 5 min at 4 °C. Carefully remove supernatant without disturbing pelleted debris. 
 

SECTION: Preparation of cell lysates
7. Quantify protein concentration and normalize across all samples. The lysate can now be subjected to the centrifugation-based solubility assay or filter trap assay described below.
SECTION: Solubility assay
8. Centrifuge lysate at 186,000 x g for 1 h at 4 °C.
SECTION: Solubility assay
9. Remove supernatant and wash pellet with 200 µL PBS.
SECTION: Solubility assay
10. Centrifuge at 186,000 x g for 30 min at 4 °C.
SECTION: Solubility assay
11. Remove PBS.
SECTION: Solubility assay
12. Add the same volume of PBS to the pellet as the lysis buffer initially used for the assay.
SECTION: Solubility assay
13. Disintegrate pellet by pipetting. Alternatively, sonication can be used.
SECTION: Solubility assay
14. Dilute total lysate, supernatant and pellet with SDS sample buffer and analyze by immunoblotting.
SECTION: Filter trap assay
15. Equilibrate cellulose acetate membrane in 0.1% SDS/H2O until completely wet.
SECTION: Filter trap assay
16. Dilute up to 200 µg (Triton) or 100 µg (RIPA) total lysate in a total volume of 200 µL pre-cooled lysis buffer.
SECTION: Filter trap assay
17. Mix samples by gentle vortexing and spin down in a mini centrifuge.
SECTION: Filter trap assay
18. Affix equilibrated membrane to a filter trap apparatus (e.g. (PR648 Slot Blot Blotting Manifold, Hoefer). Ensure that there are no bubbles trapped over the membrane.
SECTION: Filter trap assay
19. Load 200 µL 0.1% SDS/H2O to the wells and apply vacuum. Observe whether the liquid is completely drawn through.
SECTION: Filter trap assay
20. Load lysates and wait until they completely pass through the filter. 
 

SECTION: Filter trap assay
21. Wash wells 3 times with 200 µL 0.1% SDS/H2O followed by standard immunoblotting of the membrane.