Page 66 - Macromolecular Crystallography

AU TOMATION OF CRYS TALLIZATION TECHNIQUES 55

Protocol 3.8 Setting up an oil barrier over vapour diffusion trials

Equipment and reagents 5. Dispense the hanging drops on the coverslips as usual
Paraffin oil by mixing the protein solution with the reservoir solution.
Silicone oil Use the reservoir without oil as your source of precipitant
Crystallization plates for hanging and sitting drops for all the drops.
6. Invert the cover slips and place over the wells
Coverslips
containing the oil layer.
Sealing tape
7. Place the last drop over the reservoir without the oil.
Protein solution
This drop will act as your control.
Crystallization reagents
8. Incubate at the temperature of your choice.
9. Wait patiently for the results because in trials
Method for preparation of the oils containing an oil barrier, crystals require longer periods (e.g.
1. Mix paraffin and silicone oils in equal volumes. 8–10 days compared to 12–24 hours) to grow to full size
2. Shake well and allow to stand for several minutes. The but their quality is improved.
oils are totally miscible once the bubbles have disappeared. 10. If the quality of the crystals is not sufficiently improved,
repeat the protocol using different ratios of paraffin and
silicon.
Method for setting up hanging drops
1. Use a Linbro type plate for hanging drops. Method for sitting and sandwich drops
2. Coat the lips of the reservoirs with grease or oil (unless In the case of sitting and sandwich drops, set up the trials
your plates are pregreased). as you would normally do and place the layer of oil above
3. Pipette 0.6–1 ml of the reservoir solution which gives the reservoir before sealing the plates with tape (Chayen,
you the showers of crystals into each well. 2006).
4. Pipette volumes ranging from 0.1– 0.5 ml of a mixture Warning: This technique does not work with PEG or MPD
of paraffin and silicone over all the reservoirs except for concentration above 13% but is very effective at
one reservoir. The oil will form a layer above the reservoirs concentrations below 13% and at all concentrations of
(Fig. 3.6b). all salts.

(a) (b)

Crystallization Crystallization
drop drop

Barrier of paraffin
and silicone oils
Reservoir
Reservoir

Figure 3.6 Application of an oil barrier to improve crystals in hanging drops. (a) A standard hanging drop trial; (b) a trial containing an oil barrier.
Modified from Chayen (1997b). J. Appl. Cryst. 30, 198–202, with permission from the IUCr.

3.7.1 Crystallization in gelled media and sedimentation of the growing crystals, in some
way mimicking a microgravity environment (Robert
The quality of crystals can be significantly improved et al., 1999; and references therein).
when grown in gel media. Growth in gel reduces Gelled trials are performed in vapour diffusion,
both convection of the protein molecules, thus counter diffusion, and in batch. To date, the most
favouring their slow diffusion to the crystal faces, simple and speedy procedure for performing trials