Bicelle Preparation

Preparation of DHPC/DMPC-based bicelles

Materials

1,2-Dihexanoyl-sn-glycero-3-phosphocoline (DHPC, 453.507)

1,2-Dimyristoyl-sn-glycero-3-phosphocoline (DMPC, 677.933)

To make a 10 mL 15% (w/v) bicelle solution:

DHPC (453.507 g/mol)	0.2735 g
DMPC (677.933 g/mol)	1.2265 g
Phosphate buffered saline (PBS)	10 mL
TTAB (336.40 g/mol)(optional)	8.0736 mg
NaN3 (65.00987 g/mol)	2.00 mg
pH	6.5

Method

15% (w/v) solution (15 g 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 10 mM phosphate, pH 6.5, and 0.02% NaN3 buffer solution is going to be prepared.

1. Filter the deionized water before using it, because small particles tend to nucleate[1]. Prepare and weigh the correct amount of DMPC, DHPC, and TTAB, and mix with the buffer in the filtered deionized water. Do not use a sonicator to mix up the lipids.

2. Vortex the sample for 10 minutes at 4℃ and leave for 15 minutes in the cold room. The solution will become foamy and milky.

3. Vortex the sample for another minute at room temperature, and put it in a water bath at 38℃ for 30 minutes. Then put it back in the cold room for another 15 minutes.

4. Again after 30 minutes of vortexing, the sample is left to warm up to room temperature for another 15 minutes followed by another minute of vortexing and another 30 minutes in the bath at 38℃.

5. The process is repeated several times until clear, transparent fluid at a low temperature is seen. The liquid crystal phase collapses at about 45℃.

Notes

1. On Avanti note, a small amount of tetradecyltrimethylammonium bromide (TTAB) is suggested to add to the sample to increase bicelle stability. The long-term stability of bicelles is limited by the hydrolysis of the ester bonds connecting the saturated fatty acid chain and the glycerol-phosphatidylcholine head group. The hydrolysis is very dependent upon pH within a minimum of 6.5-7.0.

2. DHPC is very important to determine the bicelle dimension.

3. Use low salt concentration (< 50 mM).

4. 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 is re-heated at 38℃.

5. Two volumes of protein solution[2] are added to one volume of bicelle solution.

6. Light vortexing and heating could check if the solution is clear without particles.

7. It is suggested by Avanti, but don’t know about any knowledge about concentration.