If you look at the Phase Diagram page you'll see that to make protein crystals grow we need an initial nucleation event to occur in the so-called 'nucleation zone'. Once this happens, the effective 'soluble' protein concentration reduces as the crystal grows until the protein becomes 'undersaturated'. But how do we get that initial nucleation event? We could set up lots of screens with various protein concentrations using the microbatch technique (see the next page) in the hope that a few of the drops we set up will be in the nucleation zone from the outset.
However, the vapour diffusion technique does a lot of this work for us. The stock precipitant condition is mixed with the protein stock in a small drop (typically 0.2 -2 ul) and then allowed to equilibrate with a much larger reservoir of the condition in a sealed container. Since the small drop contains a compartively lower concentration of the precipitant (it has been diluted by the protein stock....), moisture will evaporate from the drop to the large reservoir until the precipitant concentration in both approaches equilibrium. The protein concentration in the drop effectively increases, hopefully moving through the Phase Diagram from either the Undersaturated or Metastable zones and into the Nucleation zone. If conditions are right, nucleation will take place and your protein crystal will start to grow.This technique is incredibly powerful since it screens a large area of 'crystallisation space' with each condition. You do not necessarily need to finely tune your protein concentration since equilibration will hopefully do it for you (as long as the concentration is low enough to start with....). Regularly checking your trays as
equilibrium takes place is essential!
The Department has a Cartesian robot that can very quickly set up 96-condition sitting-drop vapour diffusion experiments with numerous commercial screens. 25 ul of your protein stock is enough for one tray!