Supported Lipid Bilayer Formation

Supported lipid bilayers (SLBs) were
prepared on glass substrates (no. 1.5, Marienfeld-Superio, Lauda-Königshofen,
Germany), used for fluorescence microscopy imaging, and on silicon
wafers coated with 5 μm SiO₂ (Silicon Materials,
Kaufering, Germany), used for reflectometric interference spectroscopy
(RIfS). Both substrates were treated for 20 min with a H₂O/NH₃/H₂O₂ (5:1:1, v/v) solution
at 70 °C and subsequently activated for 30 s with O₂-plasma (Zepto LF PC, Diener electronic, Ebhausen, Germany). The
hydrophilized substrates were mounted in a measuring chamber and immediately
incubated with SUVs.
For the preparations on glass slides, SLBs
were formed by incubating the substrates for 1 h with SUVs (m = 0.2 mg, c = 0.53 mg/mL) at 20 °C
and excess lipid material was removed by a 10-fold buffer exchange
with spreading buffer followed by ezrin buffer (50 mM KCl, 20 mM Tris,
0.1 mM NaN₃, 0.1 mM EDTA, pH 7.4). For SLB formation on
silicon substrates, SUVs (m = 0.2 mg, c = 0.53 mg/mL) were spread while the optical thickness was read out.
After successful SLB formation, excess lipid material was removed
by rinsing 5 min with spreading buffer and 5 min with ezrin buffer.