Table 3. Troubleshooting table.

Step	Problem	Possible reason	Solution
5, 6	Cloudy lipid mixtures	(1) Some specific lipids have high phase-transition temperature. (2) Oxidation of unsaturated lipids	(1) Blow-dry the lipids in 45 °C water bath. (2) Purchase new lipids
8, 9	Incomplete dissolution of lipid films	Too much lipids and/or insufficient detergents in dissolution buffer	Supply more Buffer A; check the concentration of detergent in Buffer A
12, 13	No liposomes or liposome size less than 20 nm	(1) Detergents are not removed thoroughly. (2) Inappropriate detergents	(1) Decrease the volume of the samples loaded into desalting column. (2) Use suitable detergents
12, 13	Large particles (>500 nm)	Dirty cuvette	Clean the cuvette thoroughly before analysis
14, 17	Low fusion efficiency	(1) Low liposome concentration. (2) Insufficient functional SNARE proteins reconstituted on the liposomes (degradation or incorrect concentrations) (3) Non-functional SNAREs. (4) Poor lipid quality	(1) Reconfirm the concentrations of liposomes used in the assay. (2) Perform SDS-PAGE to check both the reconstituted liposomes and the SNARE stocks. Purify new proteins and add protease inhibitors during storage. Supply protease inhibitors in liposome fusion assays. (3) Check SNARE complex assembly in detergents. Try refresh the proteins or use truncated form that contains minimal SNARE domain and transmembrane domain. (4) Use freshly prepared proteoliposomes or purchase new lipids
15, 17	Negative control gives robust liposome fusion signal	Poor lipid quality	Use freshly prepared proteoliposomes or purchase new lipids