Page 67 - Macromolecular Crystallography

56 MACROMOLECULAR CRYS TALLOGRAPHY

Protocol 3.9 Preparation of the stock solutions of gels

Equipment and reagents 2. Stir the solution vigorously using a stirrer at high speed
Glass beaker or by hand. At first, a phase separation occurs which looks
Stirrer like oil drops in the solution. When stirring vigorously these
Electric device for supporting the beaker and stirrer drops disperse.
3. Top up the solution to 4 ml with deionized water.
Tetramethyl orthosilane (TMOS)
4. Stir vigorously for an additional 10–15 min keeping the
Deionized water
beaker covered. The mixture should by now have become a
Method clear solution.
Preparea4ml stock solution of TMOS at 5% (v/v) as
follows:

1. Add 0.2 ml TMOS solution to 2 ml deionized water in a
glass beaker.

Protocol 3.10 Dispensing microbatch crystallization trials in gels

Equipment and reagents 2. Fill a crystallization tray with paraffin oil as done for
Freshly made 5% (v/v) gel solution standard microbatch trials (by hand or robot).
Paraffin oil 3. Place the gel solution while it is still in liquid form into
Microbatch plates one of the channels/syringes of the dispensing system in the
same way as for the other components of the crystallization
Protein solution
mixture.
Crystallizing reagents
4. Dispense the gel solution under the oil simultaneously
Eppendorf tubes
with all the other components to form one drop.
Coverslips
5. Incubate the trials at the selected temperature(s).
Pipette 6. After a given time (12–16 hours for the gel type and
concentrations described here) polymerization occurs and
Method for manual dispensing
1. Mix the protein, crystallizing agents, buffer, etc., and the the drop gels.
freshly made gel solution in an Eppendorf tube or on a 7. Harvesting crystals from the gelled drops is done in the
coverslip. The gel solution should be at a final concentration same way as from standard microbatch trials since the gel is
of at least 0.2% (v/v). quite tenuous.
2. When mixed, draw a drop of a volume of your choice 8. Test a variety of final concentrations of the gel ranging
with a pipette tip and dispense under the oil as described in from 0.2–0.5%.
Protocol 3.1.
Gels have also been used as a slowing down mechanism
Method for dispensing by robot in various guises such as a permeable ‘plug’ between the
1. Choose a robotic dispensing system consisting of several two solutions or, in the method of gel acupuncture, as a
channels/syringes in which precipitant, buffer, protein, and barrier for the precipitant solution on its way into the
additives can be put into different syringes and dispensed protein-containing capillary (Robert et al., 1999).
simultaneously or in rapid succession.