In meso Approaches to Membrane Protein Crystallization - Molecules: Nucleation, Aggregation and Crystallization - page 266

Fig. 3. A phase diagram of monooleoyl/water system (redrawn from Cherezov et al .,

2006).

(iii) To obtain the isotropic lipidic phase, the MO is kept for an additional

20 minutes at 40 C. Subsequently, let the lipidic phase cool to room

temperature. The MO phase must remain transparent. If not, the
melting procedure should be repeated.

°

(iv) Add 1 µ l BR solution (10 mg/ml BR) with about 1.2% (w/w) of

n-octyl- β - D -glucoside (OG) per mg of MO.

(v) A homogenous lipid/protein mixture as well as the formation of

the cubic phase is achieved by the following centrifugation proce-
dure. Spin the PCR tube with the sample at 22 C at 10 000 rpm for

15 minutes. Rotate the tube within the rotor by 90

°

and spin again.
Repeat this spinning procedure four times to obtain a homogenous
mixture.

°

(vi) Leave the sample for 24 hours in the dark at 22 ° C.