In meso Approaches to Membrane Protein Crystallization - Molecules: Nucleation, Aggregation and Crystallization - page 267

(vii) Add a ground powder of KH 2 PO 4 mixed with Na 2 HPO 4 (95/5 w/w) to
obtain a final concentration of 1-2.5 M phosphate (pH 5.6).
(viii) Homogenize the sample by repeated centrifugation as described in
Step (v).

(ix) Leave the crystallization batch in the dark at 22 ° C. The first micro-

crystals of BR (10-20 m in diameter) usually appear within one

week after the phosphate has been added (Fig. 4).

µ

To summarize, BR in the buffer is mixed with MO in the FI phase.
The following procedure of centrifugation facilitates cubic phase growth
and reconstitution of BR in lipid bilayers. Crystal growth is initiated by
the addition of a precipitant — ground powder of KH 2 PO 4 mixed with
Na 2 HPO 4 . The above-mentioned protocol for crystallization is quite close
to that described in the original paper by Rosenbusch and Landau
(Landau, Rosenbusch, 1996).

(a) (b)

Fig. 4. (a) A BR crystallization probe in a PCR tube, and (b) a single BR crystal in
the probe.