Vapour Diffusion Crystallisation
Our high-throughput soluble protein crystallisation experiments are based on sitting-drop vapour diffusion geometry. The 150+150 nl droplets that we recommend are large enough so that interesting crystals may be harvested and used for diffraction experiments, making the same sitting drop plates appropriate for both initial screening and optimisation experiments.
How does vapour diffusion work? Vapour diffusion experiments rely on an initial concentration difference between the crystallant in the reservoir and the crystallant in the experimental droplet, with this concentration gradient driving water vapour (and any other volatile species) to migrate from the droplet to the reservoir. In turn, this causes the droplet to shrink, and a concomitant increase in protein concentration.
Ideally, the protein is concentrated via this process to super-saturation, and equilibrium is restored by the formation of protein crystal. Crystal formation requires the formation of a nucleus before crystal growth can continue, and nucleation is a stochastic process – both time and volume dependent. The vapour diffusion technique has been popular for decades, mostly as the ‘hanging drop’ method. The sitting drop technique is an analogous method which is used as it is easier to adapt to high-throughput technologies.
The image above compares sitting drop and hanging drop vapour diffusion methods, where the pink represents protein solution and the blue represents reservoir.
Robotic Sitting Drop
Coupled with automation, sitting drop protein crystallization methods allow very small reaction volumes to be used, as little as 100 nL of sample solution per experiment. In C3, the default (and recommended) experimental technique consist of 150 nL protein + 150 nL crystallant droplets. These droplets are equilibrated against 50 µL of crystallant in the reservoir. We set up all protein crystallisation experiments using SD-2 (IDEX Corp) plates (see image below) via nano-dispensing robots.
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