Table 1.

Troubleshooting table.

Step	Problem	Possible Reason	Possible Solution
7,17	Low fusion efficiency after Ca2+ addition for the reaction of T+VSyt1+NSF/αSNAP+M18+M13	a) Overestimated liposome concentration b) Not enough functional SNARE proteins reconstituted on the liposomes, either because of degradation or wrong concentration c) Low activity of one or more of the proteins and/or poor lipid quality.	a) Measure the liposome concentration. b) Use SDS-PAGE to check both the proteoliposomes and the SNARE protein stocks used for reconstitution. If degradation is found in the protein stocks, then purify new proteins and add protease inhibitors during storage. If degradation happened during reconstitution, make sure protease inhibitors are included. If too little protein is incorporated on the liposomes, check whether the protein stock concentration is overestimated. Prepare fresh proteins and/or buy new lipids.
21	Too much content mixing before Ca2+ addition in the reaction of T+VSyt1+NSF/αSNAP+M18+M13	Not enough EGTA	Check the concentration of EGTA. Make sure100 μM EGTA is included in the reaction.
21	Too much fusion for the control experiments T+VSyt1+NSF/αSNAP+M18, T+VSyt1+NSF/αSNAP+M13 and T+VSyt1+NSF/αSNAP	NSF is not sufficiently active	NSF can become partially inactive after storage at −80 °C. Freshly purified NSF gives the best activity.
24	Signal keeps increasing slowly after adding detergent	The detergent did not mix well with the sample	Pipette the sample up and the down at least 10 times after detergent addition to make sure the detergent is well mixed with the sample. Keep collecting the data for a few minutes, until the signal intensity becomes stable.