3 Method
Bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC) were produced with head group-functionalized lipid
(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (Biotinyl-Cap PE) or 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (PDP PE)
(18:1)). Here we describe the preparation of a 2-mol% head
group-functionalized lipid layer. The percentage of head groupfunctionalized lipid can be adjusted, but percentages above 5%
produce redundancy in the occupancy of the head groups.
3.1 Preparation
of Multilamellar
Vesicles
1. 10 mg/mL POPC stock suspension. Weigh 10 mg of lyophilized POPC within a glass bijoux with airtight lid. Dissolve in
1 mL of chloroform and swirl to homogenize.
2. 1 mg/mL head group-functionalized lipid stock suspension.
Weigh 1 mg of lyophilized Biotinyl-Cap PE or PDP PE within
a glass bijoux with airtight lid. Dissolve in 1 mL of chloroform
and swirl to homogenize (see Note 4).
3. For a 2% head group-functionalized lipid bilayer, mix 27.4 μL
of Biotinyl-Cap PE stock suspension or 23.7 μL of PDP PE
stock suspension with 96.8 μL of POPC stock suspension
within a flat bottomed glass bijoux (see Note 5).
4. Remove chloroform under a continuous gentle nitrogen
stream to produce a thin lipid film. Ensure that all chloroform
is removed by drying under vacuum overnight.
5. Rehydrate desiccated lipids in 1 mL of 0.2 μM filtered 10 mM
HEPES, 150 mM NaCl, and 2 mM CaCl 2 (pH 7.4) (final
molar concentration of 1.3 mM). Incubate at room temperature for a minimum of 30 min (see Note 6).
3.2 Lipid Extrusion
to Produce Unilamellar
Vesicles (See Note 7)
1. Assemble mini-extruder with a 50-nm-pore polycarbonate
membrane as per the manufacturer’s instructions. Ensure that
filter supports have been pre-wetted with ultrapure water or
buffer (see Note 8).
2. Pre-wet assembled extruder parts by passing 1 mL of buffer or
ultrapure water through the extruder using gastight syringes.
Discard the buffer.
3. Load hydrated lipids into gastight syringes and extrude (pass
from one syringe to the other) a minimum of 19 times (see
Notes 9 and 10).
4. Store unilamellar vesicle stock at 4
C until use, and use within
a week.
QCM-D Sensor Preparation to Study Complex Interactions
189
Bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC) were produced with head group-functionalized lipid
(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (Biotinyl-Cap PE) or 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (PDP PE)
(18:1)). Here we describe the preparation of a 2-mol% head
group-functionalized lipid layer. The percentage of head groupfunctionalized lipid can be adjusted, but percentages above 5%
produce redundancy in the occupancy of the head groups.
3.1 Preparation
of Multilamellar
Vesicles
1. 10 mg/mL POPC stock suspension. Weigh 10 mg of lyophilized POPC within a glass bijoux with airtight lid. Dissolve in
1 mL of chloroform and swirl to homogenize.
2. 1 mg/mL head group-functionalized lipid stock suspension.
Weigh 1 mg of lyophilized Biotinyl-Cap PE or PDP PE within
a glass bijoux with airtight lid. Dissolve in 1 mL of chloroform
and swirl to homogenize (see Note 4).
3. For a 2% head group-functionalized lipid bilayer, mix 27.4 μL
of Biotinyl-Cap PE stock suspension or 23.7 μL of PDP PE
stock suspension with 96.8 μL of POPC stock suspension
within a flat bottomed glass bijoux (see Note 5).
4. Remove chloroform under a continuous gentle nitrogen
stream to produce a thin lipid film. Ensure that all chloroform
is removed by drying under vacuum overnight.
5. Rehydrate desiccated lipids in 1 mL of 0.2 μM filtered 10 mM
HEPES, 150 mM NaCl, and 2 mM CaCl 2 (pH 7.4) (final
molar concentration of 1.3 mM). Incubate at room temperature for a minimum of 30 min (see Note 6).
3.2 Lipid Extrusion
to Produce Unilamellar
Vesicles (See Note 7)
1. Assemble mini-extruder with a 50-nm-pore polycarbonate
membrane as per the manufacturer’s instructions. Ensure that
filter supports have been pre-wetted with ultrapure water or
buffer (see Note 8).
2. Pre-wet assembled extruder parts by passing 1 mL of buffer or
ultrapure water through the extruder using gastight syringes.
Discard the buffer.
3. Load hydrated lipids into gastight syringes and extrude (pass
from one syringe to the other) a minimum of 19 times (see
Notes 9 and 10).
4. Store unilamellar vesicle stock at 4
C until use, and use within
a week.
QCM-D Sensor Preparation to Study Complex Interactions
189