Page 61 - Macromolecular Crystallography

50 MACROMOLECULAR CRYS TALLOGRAPHY

most suitable vessel for this method is an X-ray A microfluidic chip has been developed for
capillary, the geometry of which reduces unde- rapid screening of protein crystallization conditions
sired convective effects (García-Ruíz et al., 2002). A (Hansen et al., 2002) using the free interface diffu-
crystallization cassette has also been designed for sion method. The chip is comprised of a multilayer,
holding many capillaries that can be put into con- silicon elastomer and has 480 valves operated by
tact with various precipitant solutions for the use of pressure. The valves are formed at the intersection of
this method within a high-throughput environment two channels separated by a thin membrane. When
(Ng et al., 2003). pressure is applied to the top channel it collapses

Protocol 3.2 Fine tuning of conditions in microbatch

Equipment and reagents coverslip.) When mixed, draw the drop with a pipette tip
0.5-ml Eppendorf tubes and dispense under paraffin oil as described in Protocol 3.1.
Coverslips 2. Incubate at the temperature of your choice.
Pipette 3. Observe trials under a light microscope.
Microbatch plates
Protein solution When performed by a robot, the different ingredients are
Crystallization reagents placed in different channels/syringes of a dispensing system
and dispensed simultaneously under the oil by the action of
Paraffin oil
motorized syringes. All robots have routines whereby they
Method pick up chosen stock solutions and dispense them into a
1. Mix the protein solution and the crystallizing agents in well to which protein is added simultaneously or later on
an Eppendorf tube. (If quantities are very small, mix on a (Chayen, 2006).

Protocol 3.3 Two alternative ways of harvesting crystals from microbatch
Equipment and reagents If the above protocol proves tricky, harvest in the following
Cryoprotectant solution way:
Precipitate solution at ∼5% higher concentration than
that in the drops 1. Add harvest solution (of ∼5% higher concentration of
Micro tools (Hampton Research) precipitant than that in the drop) into the well containing
Standard pipette the crystals. If you have a 1-µl drop, add 5–10 µl of harvest
solution.
Scalpel
2. Wait a short while (up to 15 min) to allow the crystals
Loops
to equilibrate.
Depression plates
3. Withdraw the enlarged drop using a standard
10–100 µl pipette which had its tip cut off with a
Method scalpel in order to widen its bore.
1. Add a few microlitres of cryoprotectant solution to the 4. If the crystals stick to the vessel, loosen them gently
drop containing the crystals. inside the drop using micro tools or very thin strips of filter
2. After several minutes check that the crystals are not paper (the edge of the strip that will touch the crystal is best
cracked or dissolved by looking at them under the torn rather than ‘cleanly’ cut with scissors).
microscope. If they crack or dissolve, adjust the 5. Transfer the drop into a depression well containing more
concentration of cryoprotectant or change cryoprotectant. harvest solution.
3. Take the crystals directly out of the oil using a loop 6. From this stage onwards, handle the crystals as you
and freeze. would from a standard diffusion trial.