Important: Due to structural limitations that affect absorbance, the concentration of pre-formed fibrils (PFFs) cannot be calculated accurately unless they are destroyed and reduced to monomer. The multi-phase system (suspension), sonication levels and fibril length also affect the ability to measure concentration. For these reasons, the scientific literature is clear that alpha synuclein protein concentrations should be carried out prior to fibrillization.

1. 1. Measure A280 and use extinction coefficient for alpha synuclein to determine the monomer concentration.
   2. Run an SDS gel with determined concentration to check the purity and confirm concentration visibly.
   3. Move the monomer into fibrillization process.
   4. Perform sedimentation assay to ensure required amount of material has been turned into insoluble fibrils at values required for CoA (over 90%).