User Guide
HR2-320 (pg 2)
Seed Bead
TM
is a good starting point for the stabilizing solution. The simplest option is to
use the crystallization reagent (reservoir solution) that produced the crystals.
A more complex option is to perform some empirical experimentation to de-
termine the reagent composition of the stabilizing solution. The stabilizing
solution will be a reagent composition somewhere between that of the reser-
voir used to obtain the crystal and that of the drop at the initial mixing stage.
2. Remove crystals from a drop using a Mounted CryoLoop or pipet. Do not
leave the seed exposed to the air for any longer than absolutely necessary.
Macromolecular crystals have a high solvent content and can be damaged or
destroyed by evaporation of water from about the crystal.
3. Place the seed crystals in the microcentrifuge tube containing 50 micro-
liters of stabilizing solution and Seed Bead. Close the microcentrifuge tube.
4. Vortex the microcentrifuge tube containing the seed crystal and the Seed
Bead for 3 minutes. Alternatively, one may choose to sonicate the microcen-
trifuge tube containing the seed crystal and the Seed Bead for 3 minutes. See
Figure 2 below.
5. Pipet 450 microliters of the stabilizing solution into the microcentrifuge
tube containing the vortexed crystal and Seed Bead. This is your seed stock.
Preparing Serial Dilutions for Seeding
•
Dropping Solution 1:
Undiluted seed stock in stabilizing buffer.
Dilution 1x10
0
.
•
Dropping Solution 2:
45 microliters of stabilizing buffer contain-
ing no protein or crystals plus 5 microliters of Dropping Solution 1.
Dilution 1x10
-1
.
•
Dropping Solution 3:
45 microliters of stabilizing buffer contain-
ing no protein or crystals plus 5 microliters of Dropping Solution 2.
Dilution 1x10
-2
.
•
Dropping Solution 4:
45 microliters of stabilizing buffer contain-
ing no protein or crystals plus 5 microliters of Dropping Solution 3.
Dilution 1x10
-3
.
•
Dropping Solution 5:
45 microliters of stabilizing buffer contain-
ing no protein or crystals plus 5 microliters of Dropping Solution 4.
Dilution 1x10
-4
.
•
Dropping Solution 6:
45 microliters of stabilizing buffer contain-
ing no protein or crystals plus 5 microliters of Dropping Solution 5.
Dilution 1x10
-
.
Serial dilution of the seed stock can be performed if seeding experiments us-
ing the seed stock produce too many crystals in the drop. When preparing a
number of serial dilutions of the seed stock, one should reserve a portion of
each serial dilution for future crystallization experiments. What follows is an
example of how to perform a serial dilution to prepare dropping solutions
for seeding. One may prepare fewer or more dilutions depending upon how
many drops are to be set. Also, one may change the dilution from 1:10 to some
other ratio such as 1:2, 1:5, 1:20, and so on. Be certain to mix or vortex the
seed stock prior to performing each dilution. Failure to vortex mix can lead to
inconsistency. See Figure 3.
Setting the Drops - Seeding with the Seed Stock
Set sitting or hanging drops over reservoir solutions of reagent com position
identical to that used to obtain the initial seed crystals. Do not add reservoir
solution to the drops unless one wishes to further dilute the drops (Note: this
may dissolve the seeds). To slow vapor dif fusion equilibration one may dilute
the reservoir solution. To speed vapor diffusion equilibration one may use a
more concentrated res ervoir solution.
Example 1.
Original crystals grown using 2.0 M Ammonium sulfate,
0.1 M HEPES pH 6.8. Stabilizing solution is 2.0 M Ammonium sulfate,
0.1 M HEPES pH 6.8. Seed crystals from step 5 are composed of 500 ml of
2.0 M Ammonium sulfate, 0.1 M HEPES pH 6.8 and crystals, vortexed.
For the crystallization experiment, pipet 2.0 M Ammonium sulfate, 0.1 M
HEPES pH 6.8 into the reagent well (reservoir). For the drop, pipet 1 part
of protein plus 1 part of seed stock. The drop will equilibrate from 1.0 M to
2.0 M Ammonium sulfate.
Example 2.
The results of Example 1 produced numerous, small crystals
after only 24 hours. In an effort to reduce the number of crystals, increase
crystal size and slow the experiment one can reduce the concentration of the
reservoir to 70% of the original.
For the crystallization experiment, pipet 1.4 M Ammonium sulfate, 0.07 M
HEPES pH 6.8 into the reagent well (reservoir). For the drop, pipet 1 part of
protein plus 1 part of seed stock. The drop will now equilibrate from 1.0 M to
1.4 M Ammonium sulfate.
Solutions for Crystal Growth
Figure 3
Figure 2
