1. Pipette 40 μL of PFFs into a tube for micro-centrifuge (screw cap tubes are best).
2. Centrifuge at max speed for 60 minutes.
3. Carefully remove supernatant (40 μL) and transfer to a new tube. Add 10 μL of 5X Laemmli buffer and label as “supernatant”.
4. Suspend pellet in 40 μL of PBS, centrifuge again max speed for 30 minutes. Save supernatant and label as “wash”.
5. Suspend pellet in 40 μL PBS and label as “pellet”, add 10 μL 5X Laemmli buffer to this fraction.
6. Incubate at 90C for 10 minutes.
7. Load 10 μL of supernatant, wash and pellet a SDS-PAGE gel with a 15 well comb and perform SDS-PAGE. Coomassie stain and de-stain. We want to have the majority of the alpha Synuclein present in the pellet (>75%).

Note: The original protocol suggests 40 μL, however we have found 20 μL works in order to conserve on sample. Also, we will sometimes run the second spin down for 60 min rather than 30 min suggested, to ensure a better pellet. This assay is performed at the end of fibrillization, prior to diluting and aliquoting.