Optimization in Protein Crystallization
What is the best method for setting up an expansion tray in protein crystallization?
After attaining an initial crystal hit it is often necessary to setup an expansion tray. The goal of the expansion tray is to optimize the hit condition, ideally producing diffraction quality crystals. Depending on the components of the solution one needs to decide which factors to vary such as temperature, pH, the precipitating concentration, etc… Let’s say your initial hit contains:
10 % (w/v) PEG 8000 with 0.1 M CHES at pH 9.5
You decide that would you like to setup an expansion from 5 % to 15 % PEG 8000.
Method 1:
Pipette each individual component into each well. If you unsure what I mean by ‘well’ here is a picture of a hanging drop setup keeping in mind that expanding upon other setups is possible. Pipetting by hand is the most tedious approach, error prone and is limited in its ability to produce very shallow (or fine) gradients.
Method 2:
A/B gradient is based on creating two stock solutions at either end of the range you would like to expand. This method is well suited for expanding upon a single parameter, which in our example is PEG 8000.
For example the two stock solutions:
Stock A: 5 % PEG 8000 with 0.1 M CHES at pH 9.5
Stock B: 15 % PEG 8000 with 0.1 M CHES at pH 9.5
The total volume of the stocks would depend on the well volume and number of trials.
In a 12 well expansion, stock A would be pipetted into well A1 followed by a 0.1 mL decrease in subsequent wells. Stock B would start B6 and proceed reverse of stock A. Note: A1 and B6 are referring to the common grid labeling on crystallization trays
Method 3:
Four corners method is essentially expanding upon the A/B gradient by including two more stock solutions. Instead of having a start and end point there is now 4 points that overlap within the middle of the tray. This results in the ability to screen two variables in the center of tray. The figure of the expansion is quite help although the zoom feature is not.
The authors state that this approach is so new that they have yet to extensively test the technique.
Do you see simultaneously adjusting two variables in the four corner setup as advantageous? Is the four corner setup the replacement of the A/B gradient? How are you optimizing your protein crystallization trials?