Alignment Media Protocol
AIK, updated 4/8/2004:
1. DMPC/DHPC ester linkage bicelles:
2. DMPC/DHPC ether bicelles (hydrolysis resistant):
3. PEG
a. C12E5
b. C8E5
4. CPyBr
5. Gels
DMPC/DHPC ester linkage bicelles:
q=3:1
25% w/v lipid
25 g/100mL solvent. To make 1mL bicelles, need 250mg lipid.
DMPC = 687g/mol
DHPC =453.3g/mol
DMPC 3*687 = 2061
DHPC 1*453.3=453.3
2062/453.3=4.55
4.55+1=5.55
0.25/5.55=0.0450721 mol fraction
0.045*4.55 = 205 mg DMPC
0.045*1 = 45 mg DHPC
Mix each lipid separately in 500uL buffer. Make sure buffer contains D2O!! Vortex
thoroughly. DHPC dissolves quickly, DMPC is insoluble – get a white, chalky
suspension. When both solutions are homogeneous, add DHPC to DMPC and vortex
vigorously. Small, white chunks may form. Freeze with liquid N2 or acetone/dry ice
mixture (regular ice/water bath not cold enough to rapidly freeze bicelles). Thaw to RT,
vortex. Sonicate. Repeat at least 3 times and continue process until sample is clear at
RT, no chunks. If chunks remain, place bicelles in –80ºC freezer 1-2 hours, thaw, vortex.
If chunks still pleasant, try to siphon only clear, dissolved materials for bicelle use.
DMPC/DHPC ether bicelles (hydrolysis resistant):
q=3:1
15% w/v lipid
15 g/100mL solvent. To make 1mL bicelles, need 150 mg lipid.
DMPC = 649.97 g/mol
DHPC = 425.54 g/mol
3*649.97 = 1949.91
1*425.54 = 425.54
1949.91/425.54 = 4.58
4.58+1 = 5.58
0.15/5.58 = 0.02687, mole fraction
0.02687*4.58 = 123 mg DMPC
0.02687*1
= 26.9 mg DHPC
15 g/100mL = 90 mg/ 600 L
Want to make only 600 L of 15% bicelles
123*60% = 62.2 mg DMPC
26.8*60% = 12.7 mg DHPC
(This protocol was used for a real sample at pH 5.0. Aligned from 25-30c, isotropic
above and below this temperature range).
Mix each lipid separately in 300 L buffer – ensure buffer contains D2O!!!. Vortex
thoroughly. DHPC should dissolve easily. DMPC will NOT dissolve but rather forms
milky-white suspension. When both samples are homogeneous, add DHPC to DMPC,
vortex well. Small, white chunks may form. Freeze with liquid N2 or acetone/dry ice
mixture (regular ice/water bath not cold enough to rapidly freeze bicelles). Thaw to RT,
vortex. Sonicate. Repeat at least 3 times and continue process until sample is clear at
RT, no chunks. If chunks remain, place bicelles in –80ºC freezer 1-2 hours, thaw, vortex.
If chunks still pleasant, try to siphon only clear, dissolved materials for bicelle use.
DHPC: hygroscopic. Pump dry in lyophilizer before use. Alternatively, pipet some that
is stored in chloroform/methanol, remove w/ N2 and pump dry in vacuum pump (do not
use lyophilizer for organic solvents). Always allow powder samples to come to room
temp before measuring.
Vnmr (common to all LCs, may not need to adjust temp at all, may need to find temp and
temp range of alignment)
Check 2H alignment in probe. Lock, shim at 25c. Turn off lock power, recable for 2H
observe through lock channel. Make sure tn (transmitter nucleus) is set to 2H, use tpwr
~45 dB and pw ~10 us. The pw is actually MUCH longer than this, but we’re not trying
to be quantitative: we want alignment. Collect spectrum and svf. Settemp(35). Software
doesn’t know which sample to lock onto now b/c there are two lock frequencies. Don’t
bother locking and shimming unless you have a lot of time: Bo inhomogeneity shouldn’t
prevent you from seeing if your sample is aligned or not. Collect spectrum. To monitor
your sample as temp increases (and a fun way to kill time), array the parameter nt for
about 100 steps of 1 scan each. You should see the phase transition. Look for 2H
quadrupolar splittings. Measure the splittings.
If you want to check the temp range of alignment, set vttype=2 and array temp as you
would array any other parameter. Also array pad to give the sample time to equilibrate
btwn each temp. Set up the temp array and the pad array separately. Then type
array=’(pad,temp)’ and the two parameters will be arrayed simultaneously.
C12E5 (pentaethylene glycol monododecyl ether), Ruckert and Otting, JACS 122:7793
(2000). Available from Sigma.
4.25% w/w (includes mass of solvent, lipid, but NOT hexanol)
r = 0.87 (r is molar ratio C12E5:hexanol)
stable over wide pH range
Mix in this order: 25uL C12E5, 500uL water or buffer (D2O included), vortex very well.
C12E5 is viscous – do not pull the pipette out of the bottle immediately after filling
pipette tip. Wait at least 15sec to make sure C12E5 gets into pipette tip. Add 9uL
hexanol in 3uL or 4.5uL increments, vortexing well after each addition. Solution goes
from clear to milky, turbid. Then to translucent and viscous w/ lots of bubbles (upon
vortexing). When liquid crystalline, solution looks blue-ish in color. This phase does
align in the magnet but could be biphasic, heterogeneous. If solution becomes cloudy,
milky again and very fluid, you have added too much hexanol and have passed nematic
phase. Careful pipetting technique is essential!
2
H splitting at 25C should be about 23-26 Hz but may change upon addition of
biomolecule to align.
The higher the concentration of “lipid” you use, the more hexanol you may have to use.
You will have to experimentally determine the correction ration of C12E5:hexanol.
C8E5 (Pentaethylene glycol octylether)
5% w/w (includes mass of solvent, lipid, NOT octanol)
r = 0.84 (r = molar ratio C8E5:octanol)
(C8E5 more soluble in water than C12E5)
e.g. 5% C8E5 w/ r=0.84 aligns at 25C to 24-26Hz 2H quad splitting
Add 176 uL C8E5 to Eppendorf tube. Add 640 uL buffer (this buffer does not have
D2O), 100 uL D2O, 64 uL octanol in 8 uL increments.
Another C8E5 protocol that also works:
51uL C8E5, 16uL octanol, 100uL D2O (or if D2O already in buffer ignore this step and
add 100uL to buffer amount), 825 uL buffer. 4.83w/w% C8E5, r=1.35. 2H splits to
32Hz.
CPBr (cetylpyridinium bromide) Barrientos et al, JBNMR 16:329-337 (2000).
6.5% (w/v) Cetylpyridinium bromide
25 mM NaBr
hexanol
r, CPBr:hexanol, = 1:1.33 (w/w)
Stable over wide pH range