Final concentration	Volume to add (μl)
HEPES buffer 1		36.4
TCEP (10 mM)	0.5 mM	10.0
Mg2+ (50 mM)	2.5 mM	10.0
T-liposomes (3.2 mM)	250 μM	15.6
ATP (200 mM)	2 mM	2.0
αSNAP (50 μM)	2 μM	8.0
NSF (20 μM)	0.8 μM	8.0
Munc18–1 (20 μM)	1 μM	10.0
EGTA (10 mM)	100 μM	2.0
Streptavidin (100 μM)	5 μM	10.0
Incubate the components above at 37 °C for 25 min
HEPES buffer 2		77.5
V-Syt1 liposomes (2 mM)	125 μM	12.5
Munc13–1 C1C2BMUNC2C (20 μM)	0.5 μM	5.0
SNAP-25 (40 μM)	1 μM	5.0
Transfer 190ul to cuvette for data acquisition.
CaCl2 (10 mM) at 5min	0.5 mM	10.0
βOG (20 %) at the end	1%	10.0