3. EZ-Link Maleimide-PEG n -biotin reagent is moisture sensitive.
Upon use, allow the reagent to reach room temperature inside
the packaging with desiccant before opening to avoid moisture
condensation.
4. Stock suspensions of lipids in chloroform can be stored under
nitrogen within airtight glass containers at À20
C for approximately 3 months. To observe if evaporation has occurred during storage, make a mark on the outside of the glass container
at the chloroform meniscus. Evaporation will lead to a change
of lipid concentration within the solution, and volumes of
lipids required in subsequent steps will need to be adjusted
accordingly.
5. Alternative percentage concentrations of functionalized lipids
can be used. However, concentrations over 5% do not lead to
greater mass coverage of avidin and lead to redundancy of the
linkers. Molar percentage greater than 10% can be detrimental
to bilayer formation.
6. If alternative lipids are to be used, consideration must be given
to the phase transition temperature (T m ) of the lipid. Rehydration must be performed at temperatures above the T m of the
lipid.
7. Unilamellar vesicles can also be produced from rehydrated
multilamellar vesicle solutions using sonication. Investigation
would be required to achieve the ideal sonication strength and
length for individual sonicators to produce correctly sized unilamellar vesicles. Consideration of the T m of the base lipid
(nonfunctionalized lipid) may also limit the use of sonication.
Sonication must be performed at temperatures above the T m ;
therefore, sonicating on ice to prevent overheating and evaporation may not be achievable.
8. If alternative lipids are to be used, consideration must be given
to the T m of the base lipid (nonfunctionalized lipid). Extrusion
should be performed at a temperature above the T m of the base
lipid. This can be achieved by placing the mini-extruder on a
hot plate and allowing approximately 15 min for the extruder
to reach the required temperature before use.
9. Use the same syringe used to take up buffer in the previous step
to load lipid sample. It is also important to always pass lipids
through the membrane an odd number of times. This ensures
that any unwanted particulates are filtered by the membrane
and are not in the final extruded lipid solution. Lipid suspension should change from cloudy to clear following extrusion.
10. Unilamellar vesicle size can be checked using dynamic light
scattering. Vesicles should be ~80 nm and homogeneous when
extruded through a 50-nm pore size filter. Vesicles considerably below this size should be discarded. If vesicles are larger,
QCM-D Sensor Preparation to Study Complex Interactions
193
Upon use, allow the reagent to reach room temperature inside
the packaging with desiccant before opening to avoid moisture
condensation.
4. Stock suspensions of lipids in chloroform can be stored under
nitrogen within airtight glass containers at À20
C for approximately 3 months. To observe if evaporation has occurred during storage, make a mark on the outside of the glass container
at the chloroform meniscus. Evaporation will lead to a change
of lipid concentration within the solution, and volumes of
lipids required in subsequent steps will need to be adjusted
accordingly.
5. Alternative percentage concentrations of functionalized lipids
can be used. However, concentrations over 5% do not lead to
greater mass coverage of avidin and lead to redundancy of the
linkers. Molar percentage greater than 10% can be detrimental
to bilayer formation.
6. If alternative lipids are to be used, consideration must be given
to the phase transition temperature (T m ) of the lipid. Rehydration must be performed at temperatures above the T m of the
lipid.
7. Unilamellar vesicles can also be produced from rehydrated
multilamellar vesicle solutions using sonication. Investigation
would be required to achieve the ideal sonication strength and
length for individual sonicators to produce correctly sized unilamellar vesicles. Consideration of the T m of the base lipid
(nonfunctionalized lipid) may also limit the use of sonication.
Sonication must be performed at temperatures above the T m ;
therefore, sonicating on ice to prevent overheating and evaporation may not be achievable.
8. If alternative lipids are to be used, consideration must be given
to the T m of the base lipid (nonfunctionalized lipid). Extrusion
should be performed at a temperature above the T m of the base
lipid. This can be achieved by placing the mini-extruder on a
hot plate and allowing approximately 15 min for the extruder
to reach the required temperature before use.
9. Use the same syringe used to take up buffer in the previous step
to load lipid sample. It is also important to always pass lipids
through the membrane an odd number of times. This ensures
that any unwanted particulates are filtered by the membrane
and are not in the final extruded lipid solution. Lipid suspension should change from cloudy to clear following extrusion.
10. Unilamellar vesicle size can be checked using dynamic light
scattering. Vesicles should be ~80 nm and homogeneous when
extruded through a 50-nm pore size filter. Vesicles considerably below this size should be discarded. If vesicles are larger,
QCM-D Sensor Preparation to Study Complex Interactions
193