Table 2 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
10 (Procedure 1), 11 (Procedure 2)	Blank fluorescence reading	The instrument does not have the right filters	Replace the filters to 435Ex/485Em, 450Ex/480Em or similar
		The reading optics do not match the type of plate The plate sealer is not transparent at the needed wavelength	Clear-bottom plates are compatible with top and bottom optics, but black-bottom plates are compatible only with top reading Use the recommended plate seal (cat. no. 4311971) or use a clear- bottom plate
16 (Procedure 1), 17 (Procedure 2)	Spontaneous self-aggregation	PIPES buffer	Prepare new PIPES and use NaOH only for pH adjustment. If too much NaOH is added, start again and prepare a new solution. Do not add HCl or other acids
		Undissolved PIPES	Pass the buffer through 0.22-μm filters to remove undissolved PIPES
		Hydrated PIPES powder	Buy new reagent and maintain it inside a desiccator cabinet
		Substrate contains pre-aggregated αSyn	Re-filtrate the substrate by using a 100-kDa-cutoff filter
		A shaking mode other than orbital shaking was selected	Program the shaker for orbital shaking. Protocols that use double orbital shaking use different shaking speeds and overall assay conditions
		Poor-quality substrate	Confirm the protein concentration of the stock. Underestimation could lead to a higher concentration of the protein in the assay than desired Buy or purify a new batch of substrate
		Substrate dimerization	Make sure that the plasmid encodes tyrosine at codon 136 by using TAT nucleotides70 Analyze the substrate by using DTT or 2-Mercaptoethanol (BME). If dimers were formed by disulfide bonds, after DTT, the protein should appear monomeric. If this is the problem, the plasmid needs to be modified as above If the dimers remain (confirmed by western blot), get a new batch of rec-αSyn