4. Prepare 0.13 mM unilamellar lipid solution for bilayer deposition. Dilute 100 μL of unilamellar vesicle stock with 900 μL of
running buffer in a 1.7 mL tube and mix by pipetting.
5. Temporarily stop the pump (stopping the pump between injections is not necessary in the Omega system as flow is continuous). Move the inlet tube to the unilamellar lipid solution in
the 1.7-mL tube. Ensure that no air bubbles are present within
the tubing. Change the pump speed to 25 μL/min and restart
the pump.
6. Lipid bilayer deposition should give the characteristic signal as
shown in Fig. 1 when measuring in the seventh overtone (see
Note 19). Fully formed bilayers should produce final frequency and dissipation values of approximately À25 Hz and
0.1 Â 10
À6
, respectively (see Fig. 1).
7. Once bilayer has formed and signals have stabilized, stop the
pump. Move the inlet tube back into the running buffer ensuring that no bubbles are present in the tubing. Change the
pump speed to 100 μL/min and restart the pump. Allow the
running buffer to flush through the system to ensure removal
of free lipid (approximately 10 min). If preparing a BiotinylCap PE surface, proceed to step 14 (see Notes 20 and 21).
8. Prepare 10 mM TCEP by diluting 40 μL of TCEP stock in
960 μL of running buffer in a 1.7-mL tube.
9. Stop the pump. Move the inlet tube to the TCEP solution in
the 1.7-mL tube. Ensure that no air bubbles are present within
the tubing. Change the pump speed to 50 μL/min and restart
the pump. Pump TCEP solution over the lipid surface for a
minimum of 10 min or until the response has plateaued.
10. Stop the pump. Move the inlet tube back into the running
buffer ensuring that no bubbles are present in the tubing.
Change the pump speed to 100 μL/min and restart the
pump. Allow the running buffer to flush through the system
to ensure removal of free TCEP (approximately 10 min).
11. Prepare biotin linkers. Dilute 5 μL of 200 mM EZ-Link maleimide-PEG 2 -biotin stock/200 mM EZ-Link maleimidePEG 11 -biotin stock in 995 μL of running buffer to give
1 mM final concentration.
12. Stop the pump. Move the inlet tube to the linker solution in
the 1.7-mL tube. Ensure that no air bubbles are present within
the tubing. Change the pump speed to 50 μL/min and restart
the pump. Pump linker solution over lipid surface for a minimum of 10 min or until the response has plateaued.
13. Stop the pump. Move the inlet tube back into the running
buffer ensuring that no bubbles are present in the tubing.
QCM-D Sensor Preparation to Study Complex Interactions
191
running buffer in a 1.7 mL tube and mix by pipetting.
5. Temporarily stop the pump (stopping the pump between injections is not necessary in the Omega system as flow is continuous). Move the inlet tube to the unilamellar lipid solution in
the 1.7-mL tube. Ensure that no air bubbles are present within
the tubing. Change the pump speed to 25 μL/min and restart
the pump.
6. Lipid bilayer deposition should give the characteristic signal as
shown in Fig. 1 when measuring in the seventh overtone (see
Note 19). Fully formed bilayers should produce final frequency and dissipation values of approximately À25 Hz and
0.1 Â 10
À6
, respectively (see Fig. 1).
7. Once bilayer has formed and signals have stabilized, stop the
pump. Move the inlet tube back into the running buffer ensuring that no bubbles are present in the tubing. Change the
pump speed to 100 μL/min and restart the pump. Allow the
running buffer to flush through the system to ensure removal
of free lipid (approximately 10 min). If preparing a BiotinylCap PE surface, proceed to step 14 (see Notes 20 and 21).
8. Prepare 10 mM TCEP by diluting 40 μL of TCEP stock in
960 μL of running buffer in a 1.7-mL tube.
9. Stop the pump. Move the inlet tube to the TCEP solution in
the 1.7-mL tube. Ensure that no air bubbles are present within
the tubing. Change the pump speed to 50 μL/min and restart
the pump. Pump TCEP solution over the lipid surface for a
minimum of 10 min or until the response has plateaued.
10. Stop the pump. Move the inlet tube back into the running
buffer ensuring that no bubbles are present in the tubing.
Change the pump speed to 100 μL/min and restart the
pump. Allow the running buffer to flush through the system
to ensure removal of free TCEP (approximately 10 min).
11. Prepare biotin linkers. Dilute 5 μL of 200 mM EZ-Link maleimide-PEG 2 -biotin stock/200 mM EZ-Link maleimidePEG 11 -biotin stock in 995 μL of running buffer to give
1 mM final concentration.
12. Stop the pump. Move the inlet tube to the linker solution in
the 1.7-mL tube. Ensure that no air bubbles are present within
the tubing. Change the pump speed to 50 μL/min and restart
the pump. Pump linker solution over lipid surface for a minimum of 10 min or until the response has plateaued.
13. Stop the pump. Move the inlet tube back into the running
buffer ensuring that no bubbles are present in the tubing.
QCM-D Sensor Preparation to Study Complex Interactions
191