Table 3. Troubleshooting guide for α-Syn purification issues.

Problem	Potential causes	Solutions
Low purity after anion exchange chromatography	(1) Overloading column (2) Insufficient lysate clarification	(1) Load ≤10 mg total protein per run (2) Centrifuge lysate at 40,000g for 1 h before loading
Protein aggregation during size exclusion chromatography (SEC)	(1) High protein concentration (2) Improper buffer pH or contaminants	Dilute the sample to ≤2 mg/mL before SEC
Low yield of purified α-Syn	(1) Inefficient lysis (2) Loss of protein during purification steps	(1) Ensure complete bacterial lysis using 4–5 cycles of high-pressure homogenization (2) Pool fractions containing α-Syn to maximize yield