Page 60 - Macromolecular Crystallography

AU TOMATION OF CRYS TALLIZATION TECHNIQUES 49

3.4.4 Crystallization of membrane proteins some practice it can be achieved easily (Chayen,
in microbatch 1998, 2006).
An increasing number of membrane proteins, in a
variety of different detergents, have been crystal- 3.4.6 Diffusion techniques
lized in microbatch under oil. Some of these had
failed to crystallize by all methods other than micro- Although microbatch is the simplest method of
batch. Dispensing is quick and simple, even when crystallization, it is a relatively new technique and
performed manually, and the drops in oil do not many experimenters still prefer to use vapour dif-
spread out as they do in vapour diffusion over the fusion which has been around and has worked
siliconized coverslips (Chayen, 2006). Using robots well for over 40 years. Hence, there has also been
thousands of microbatch trials can be dispensed in major development in automating and scaling
high-throughput mode in nanolitre volumes. down the quantities of sample using the popular
The protocol for setting up microbatch experi- vapour diffusion method (both sitting and hanging
ments containing membrane proteins is identical to drops). An increasing variety of robots are available
that described in Protocols 3.1 and 3.2. commercially.
The liquid–liquid free interface diffusion (FID)
method, in which protein and precipitant solutions
3.4.5 Harvesting and mounting crystals from
are carefully superimposed and left to slowly mix
microbatch
diffusively, was least used in the past due to hand-
Harvesting crystals from microbatch is slightly more ling difficulties. However, in the last 4 years the
difficult than harvesting from coverslips or from free interface technique has experienced a revival
standard sitting drops (Protocol 3.3). However, after for both screening and optimization procedures. The

Protocol 3.1 Setting up a screen in a microbatch experiment manually
Equipment and reagents when pressing them. Dispense the drop into the oil while
Screening solutions from commercial or home made kits holding the pipette on the first stop; otherwise you will
Protein solution introduce air bubbles into the drop.
Pipette of 1–2 µl 4. Add 1 µl of protein solution to the same well in the
same way. The two (separate) 1-µl drops join and become
Optional: automated hand held pipette
a2-µl drop. If the drops don’t coalesce, mix them gently
Light microscope
with the pipette tip.
Paraffin oil (Hampton Research or Molecular Dimensions)
5. Incubate at the temperature of your choice.
Silicone oil (Hampton Research or Molecular Dimensions)
6. Observe trials regularly under a light microscope.
Microbatch plates (e.g. Nunc,Terazaki, Douglas
Instruments) Method for setting up and using a robot
There are several robots for setting up screening
Method experiments in microbatch (e.g. Luft et al., 2003; Chayen
1. Pipette or pour 6 ml of paraffin oil into a microbatch et al., 1992; DeLucas et al., 2003). The precipitant solutions
plate. The oil will spread over the plate and cover the wells. are transferred simultaneously from stock screening
2. Withdraw 1 µl of the screen solution from its container solutions to crystallization plates by any number of syringes,
using a pipette. depending on the robotic system. The drops are dispensed
3. Insert the pipette tip into the well under the surface of under oil and the protein is added to the precipitant
the oil and dispense the 1 µl drop onto the floor of the drops using a dedicated syringe for the protein solution,
plate. As you withdraw the tip from the oil, the drop will either simultaneously with the screening solutions or at a
detach from it and fall to the bottom of the well within a later stage. Some of the robots have a routine of mixing
few seconds (Fig. 3.2). Pipettes usually have two stops the drops.