The detailed protocol for crystallizing membrane proteins in LCP was published by Martin Caffrey and Vadim Cherezov in: Crystallizing Membrane Proteins Using Lipidic Mesophases, Nat. Protoc. 2009; 4(5): 706–731.
More information about LCP can be found here:
> Vadim Cherezovs USC web site “The Cherezov Lab”
> CSIROs Collaborative Crystallisation Centre web site “Membrane Proteins & the Lipidic Cubic Phase”
PCC’s standard procedure for preparing the mesophase follows closely the method described by Martin Caffrey and Vadim Cherezov by mixing two parts protein solution (40%) with three parts monoolein (60%; CAS# [111-03-5]; HR2-435). For this procedure two Hamilton syringes (25µL, 50µL or 100µL Syringe Model 1710 RN) get connected by a LCP syringe coupler (Formulatrix, SKU 209526). The mixing process by moving the syringe plungers back and forth is repeated until the cubic mesophase becomes homogenous, clear and does not show any significant birefringence. The protein sample volume required for this procedure is listed in Sample Volume.
The Gryphon-LCP dispenses a 40nl bolus of the LCP-protein mixture and an excess of 800nl precipitant solution in each of the 96 well of a LCP-sandwich plate. The volume of the LCP bolus range between 20nl – 200nl. The SWISSCI LCP sandwich plate used for all LCP-experiments consists of a hydrophobic SBS sized base plate with a 60um or 120um spacer. The experiments are sealed with a glass- or a Laminex Film (MD11-54) cover. PCC recommends for bolus volumes <50nl the sandwich plates with the 60um spacer and the glass cover. The LCP-plate will be incubated at 20°C in the Rock Imager
A detailed instruction for this protein crystallization technique is posted on JoVE_Protocol_3383. by Rachna Ujwal & Jeff Abramson including the corresponding video article:"High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method".
The preparation of the protein-bicelle mixture is the responsibility of the researcher.
Pre-mixed bicelle products can be purchased through "Anatrace" or through "MemX Biosciences" / "Molecular Dimensions".
The Gryphon-LCP is dispensing in each 96 well of a VD-plate 50nl of the protein-bicelle mixture and 800nl precipitant solution. The bicelle-plate can be set up and incubated at either 4°C or 20°C in the Rock Imager. The images are taken according to the “LCP-1” imaging schedule listed in the previous section.