DMPC and DHPC phospholipids can be purchased as dry powders or already prepared mixture at different phospholipids molar ratios from Avanti Polar Lipids Inc. and used without further purification. They can be stored in a freezer at -18 deg C.

A small amount of tetradecyltrimethylammonium bromide (TTAB) can be added to the sample to increase bicelle stability (it can be purchased from Sigma).

A 15% w/v (15 g of total lipids in 100 ml) solution of lipids with a DMPC/DHPC ratio of 3:1 (mol:mol), 2.4 mM of TTAB in a phosphate 10 mM, pH = 6.5, and 0.02% NaN3 buffer solution was prepared in the following way:

• DHPC (the waxy one), which is very important to determine bicelle dimension, is particularly hygroscopic and hydrolyses slowly in the presence of water. Dry the freshly bought lipids is an optional if the storage packages are opened straight within the glass box.

• Use a glass box under N2 atmosphere to weigh the proper amounts of DHPC, DMPC and TTAB into three eppendhorf cups.

• The three compounds are dissolved together with 4 ml of buffer solution. It seems there is a connection between the source of deionised water used for the buffer and bicelle formation. Filter deionised water before using it.
  After 10 minutes of vortexing at 4 deg C the sample is left for 15 minutes in the cold room. The solution is now foamy and milky and the lipids are not completely dissolved. Therefore after another 1 minute of vortexing at room temperature the sample is put in a water bath at 38 deg C for 30 minutes and then back in the cold room for other 15 minutes to cool down. Again after 30 minutes of vortex the sample is left to warm up to room temperature for other 15 minutes followed by another minute of vortexing and other 30 minutes in the bath at 38 deg C.

• The process is repeated several times and eventually the following changes are observed: the solution is clear, transparent and fluid at low temperature. At about 25 deg C it becomes white and milky and then at 38 deg C the solution is again clear and transparent but very viscous. (People can see a blue taint in the solution). The liquid crystal phase collapses at about 45 deg C.

• At this stage the volume of the solution can be adjusted adding the appropriate volume of buffer solution. No changes is observed in the behaviour.

Warnings

• Do not use ultrasound to mix up lipids;

• Before adding the protein test the stability of bicelles in the magnet;

• Use low salt concentration (< 50mM);

• Use NaN3 for bugs, they love that stuff;

• Make sure solutions do not contain small particles, these tend to nucleate disaster.
  Before using them (the lipids separate after about a week) do a light vortexing and heating again until the phase is clear;

• Proteins which tend to aggregate, to interact with lipids, that have hydrophobic patches on their surfaces, that are partially unfolded at the bicellar transition point may cause an instability in the nematic phase which can affect the measurements.

The long term stability of bicelles is limited by the hydrolysis of the ester bonds connecting the saturated fatty acid chains and the glycerol-phosphatidylcholine headgroup.
This hydrolysis is catalised both by base and by acid, and is therefore a strong function of the pH, within a minimum at 6.5-7.0, this justifies the pH choice. At this latter pH the solution is stable for months at lower or higher pH the bicelle stability makes the sample last for less than one day.
There will be a slower decrease in the transition temperature as the sample ages resulting from a more effective hydrolysis of DHPC relative to DMPC.
Samples may be stored frozen, refrigerated or at room temperature with no noticeable effect on the reproducibility of the liquid crystalline phase once the sample are reheated at 38 deg C. However frozen sample hydrolyses much more slowly so it is better to store them at -80 deg C where they can last for months.

Diluite liquid crystalline NMR samples can be obtained by adding buffer to the 15% stock solution followed by vortexing and a light centrifugation.

The deuterium solvent signal, normally used for field-frequency lock purposes, provides a very convenient probe for monitoring the liquid crystalline state. There is a microscopic phase separation as evidenced by the simultaneous presence of a 2H singlet and a 2H doublet as the temperature raises.
Above 38 deg C the 2H solvent signal shows a well defined doublet splitting, with very narrow line widths as a consequence of the rapid exchange of water molecules between partially aligned hydration shell of oriented bicelles and the bulk solvent resulting in an incomplete averaging of the large 2H quadrupole coupling.
Insertion of the sample into the heated probehead into the magnet makes the 2H doublet appear within few seconds.
The 2H splitting is, to a good approximation, proportional to the bicelles concentration.

Above a given threshold, there is no significant effect of the magnetic field strength on the degree of alignment and no difference in solvent 2H splitting or protein alignment is detected between measurements at 500 and 600 MHz.

The small degree of alignment which results from the protein's diamagnetic susceptibility, typically is negligible relative to the much larger alignment induced by the liquid crystal.

Besides density effect a small change in the temperature across the sample can effect the splitting width of 2H and this can influence when shimming the sample.

To test the system the deuterium splitting can measured after hours or monitored overnight.

If no meaningful change in both the magnitude and line widths of the splitting are observed no phase separation has occurred and bicelles are stable. The asymmetry in the doublet intensity observed after shimming can be used as a sensitive indicator for monitoring the homogeneity of the liquid crystal phase.

A concentration of bicelles of 3.5 % (w/v) produces a splitting of about 6 Hz in the deuterium line. This should give values for the magnitude of the molecular alignment tensor of about 0.001.

For alignment significantly larger than this value the observed 1H signals will increase considerably in line width as a result of the multitude of residual 1H-1H dipolar interactions. Therefore alignment values of 0.001 are optimal for the purpose of extracting heteronuclear couplings in fully protonated proteins.

Higher degree of alignment can also result in poor INEPT magnetisation transfer for spin pairs where the dipolar contribution is comparable in magnitude to the one-bond J coupling.