Why should I work with diluted or undiluted screens?

Two approaches are used with regards to screening solution. One involves use of undiluted screens and the other uses diluted screens. The two approaches will be described in greater detail in the protocol section of the NeXtal CubicPhase Handbook.

[A] Undiluted screens are added to the protein well of the crystallization plates in a 1:1 ratio (e.g., 100 nl of screening solution + 100 nl of protein solution in the protein well). This is the traditional protocol used for most vapor diffusion experiments. However for in meso phase crystallization, other factors have to be considered, as this is a dynamic system which goes through several phases as described in the NeXtal CubicPhase handbook. The phases depend on the ratio between aqueous solution and lipids (MO). When the experiment starts, there is an excess of aqueous solution (86% aqueous solution vs. 14% lipids), this is the sponge phase.

[B] Crystallization in the sponge phase is possible, however to exploit the full power of in meso crystallization, other phases (cubic phase and lamellar phase) should also be considered. This is possible only when diluted screening solution is added directly to the protein well. This leads to further reduction of the volume upon equilibration and to an increase of protein concentration beyond the initial value. Depending on the dilution factor, this also results in different final hydration levels, hence different lipid structures (e.g., cubic phase and lamellar phase). The dilution of the screening solution is an experimental factor that needs to be optimized. As a starting point we recommend to use 1/1; 1/4; 1/8 dilutions.

Example: - 100 nl protein solution + 100 nl undiluted screen => Sponge phase - 100 nl protein solution + 100 nl diluted screen => Sponge phase => CubicPhase => Lamellar Phase (dehydration).






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