Material and Methods
Assay conditions
SNARE proteins were expressed and purified as
described (Schuette et al. 2004). Proteoliposomes
used in this experiment were prepared as
described in 20 mM HEPES, pH 7.4, with 150 mM
KCl (Pobbati et al. 2006). Lipid composition was in
5:2:2:1 ratio of brain L-α-phosphatidylcholine, L-α-
phosphatidylethanolamine (PE), L-α-
phosphatidylserine and cholesterol (Avanti).
Protein:lipid ratio was 1:4000.
For the experiment described here, a 20 mM
HEPES, 150 mM KCl at pH 7.4 buffer has been
used. The measurement was performed in
standard treated capillaries (Cat#K002).
Instrumentation
The measurements were done on NanoTemper
Monolith NT.015 and NT.115 instruments.
The measurement was performed at 40 % LED
and 40 % MST power, Laser-On time was 30 sec,
Laser-Off time 5 sec.
References
Schütte et al., Determinants of liposome fusion mediated by
synaptic SNARE proteins.
Proc Natl Acad Sci USA 2004; 101;2858–2863.
Pobbati et al.,
N- to C-terminal SNARE complex assembly promotes rapid
membrane fusion
Science 2006 313;673–676
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