Chalfont, UK). Mini-extruder set was purchased from Avanti
Polar-Lipids (Alabaster, USA). QCM-D measurements were performed on a Q-sense E1 with a standard flow module or Omega
automated system with the 8-channel standard flow module
(Q-sense Ltd., Gothenburg, Sweden). QCM-D SiO 2 coated sensor
crystals were also purchased from Q-sense Ltd. All QCM-D measurements were undertaken in flow mode (continuous solution
delivery to the measurement chamber) using a peristaltic pump in
the case of the E1.
2.1 Buffers
l
100 mM CaCl 2 . Weigh 1.1 g of CaCl 2 (anhydrous) and add to a
500-mL graduated cylinder or beaker. Add ultrapure water to
100 mL and mix using a stir bar (see Note 1).
l
10 mM HEPES, 150 mM NaCl, 2 mM CaCl 2 , pH 7.4. Add
approximately 100 mL ultrapure water to a 1-L graduated cylinder or beaker with a stir bar. Weigh 2.38 g of HEPES and
transfer to the cylinder. Weigh 8.77 g of NaCl and transfer to the
cylinder. Add ultrapure water to a volume of 900 mL and mix.
Add 20 mL of 100 mM CaCl 2 and mix. Adjust to pH 7.4 using
NaOH and then add ultrapure water to a final volume of 1 L.
Filter using a 0.2-μm membrane and degas. Buffer can be stored
at room temperature for 1 week (see Note 2).
2.2 Proteins
l
1 mg/mL avidin/streptavidin. Weigh 1 mg of avidin/streptavidin into a 1.7-mL tube. Add 1 mL of 10 mM HEPES,
150 mM NaCl, and 2 mM CaCl 2 (pH 7.4) and vortex to mix.
Syringe filter at 0.2 μM. Store at 4
C for up to 1 week. Alternatively, aliquot and freeze at À20
C.
2.3 Reagents
l
200 mM EZ-Link maleimide-PEG 2 -biotin stock. Weigh
25 mg of EZ-Link maleimide-PEG 2 -biotin into a 1.7-mL
tube. Add 238 μL of DMSO and mix by pipetting. Aliquot
and freeze at À20
C. Aliquots will be stable for approximately
3 months (see Note 3).
l
200 mM EZ-Link maleimide-PEG 11 -biotin stock. To 25 mg
EZ-Link maleimide-PEG 11 -biotin, add 136 μL of DMSO and
mix by pipetting. Aliquot and freeze at À20
C. Aliquots will be
stable for approximately 3 months.
l
250 mM tris(2-carboxyethyl)phosphine (TCEP). Weigh
71 mg of TCEP and dissolve in 1 mL of ultrapure water in a
1.7-mL tube. Mix by pipetting and aliquot. Aliquots can be
stored at À20
C for approximately 6 months.
l
2% sodium dodecyl sulfate (SDS) (w/v). Weigh 2 g of SDS
and dissolve in 100 mL of ultrapure water. Store at room
temperature.
188
Holly L. Birchenough and Thomas A. Jowitt
Polar-Lipids (Alabaster, USA). QCM-D measurements were performed on a Q-sense E1 with a standard flow module or Omega
automated system with the 8-channel standard flow module
(Q-sense Ltd., Gothenburg, Sweden). QCM-D SiO 2 coated sensor
crystals were also purchased from Q-sense Ltd. All QCM-D measurements were undertaken in flow mode (continuous solution
delivery to the measurement chamber) using a peristaltic pump in
the case of the E1.
2.1 Buffers
l
100 mM CaCl 2 . Weigh 1.1 g of CaCl 2 (anhydrous) and add to a
500-mL graduated cylinder or beaker. Add ultrapure water to
100 mL and mix using a stir bar (see Note 1).
l
10 mM HEPES, 150 mM NaCl, 2 mM CaCl 2 , pH 7.4. Add
approximately 100 mL ultrapure water to a 1-L graduated cylinder or beaker with a stir bar. Weigh 2.38 g of HEPES and
transfer to the cylinder. Weigh 8.77 g of NaCl and transfer to the
cylinder. Add ultrapure water to a volume of 900 mL and mix.
Add 20 mL of 100 mM CaCl 2 and mix. Adjust to pH 7.4 using
NaOH and then add ultrapure water to a final volume of 1 L.
Filter using a 0.2-μm membrane and degas. Buffer can be stored
at room temperature for 1 week (see Note 2).
2.2 Proteins
l
1 mg/mL avidin/streptavidin. Weigh 1 mg of avidin/streptavidin into a 1.7-mL tube. Add 1 mL of 10 mM HEPES,
150 mM NaCl, and 2 mM CaCl 2 (pH 7.4) and vortex to mix.
Syringe filter at 0.2 μM. Store at 4
C for up to 1 week. Alternatively, aliquot and freeze at À20
C.
2.3 Reagents
l
200 mM EZ-Link maleimide-PEG 2 -biotin stock. Weigh
25 mg of EZ-Link maleimide-PEG 2 -biotin into a 1.7-mL
tube. Add 238 μL of DMSO and mix by pipetting. Aliquot
and freeze at À20
C. Aliquots will be stable for approximately
3 months (see Note 3).
l
200 mM EZ-Link maleimide-PEG 11 -biotin stock. To 25 mg
EZ-Link maleimide-PEG 11 -biotin, add 136 μL of DMSO and
mix by pipetting. Aliquot and freeze at À20
C. Aliquots will be
stable for approximately 3 months.
l
250 mM tris(2-carboxyethyl)phosphine (TCEP). Weigh
71 mg of TCEP and dissolve in 1 mL of ultrapure water in a
1.7-mL tube. Mix by pipetting and aliquot. Aliquots can be
stored at À20
C for approximately 6 months.
l
2% sodium dodecyl sulfate (SDS) (w/v). Weigh 2 g of SDS
and dissolve in 100 mL of ultrapure water. Store at room
temperature.
188
Holly L. Birchenough and Thomas A. Jowitt