Granada Crystallization Box
(Pincha aquí para la versión en castellano).
After several years exploring the possibility of the coupling between
reaction (or crystallization) and diffusion, we have developed and produced a
simple crystallization device to facilitate its practice in the laboratories. The
box, named Granada Crystallization Box (GCB) consists of 4 elements made of polystyrene:
- A
reservoir to introduce the gel
- A
guide to hold capillaries
- A
cover
- A
holder to maintain the boxes
The GCB has been designed to be used in four different ways:
- To
grow crystals inside gels under diffusion controlled mass transport.
- To
grow protein crystals inside capillaries with un-gelled precipitating
agent by the counter-diffusion technique.
- To
grow protein crystals inside capillaries with gelled precipitating agent
by the counter-diffusion technique.
- To
grow protein crystals inside capillaries by the batch method.
Download video animation
GCB is commercialized by Hampton
Research ( www.hamptonresearch.com
) and Triana Science & Technology ( www.trianatech.com)
How to use the GCB
by Juan Ma. Garcia-Ruiz
A) Growth of crystals inside gels under diffusion
controlled mass transport.
The growth of crystals inside
gels is very simple. Below you can find three different recipes.
A.1. Crystallization of a slightly soluble compound by
means of a chemical reaction provokes in most cases a cloud of small
crystallites or amorphous precipitate. The use of gels solves the problem in
most cases provided that the reactants are soluble in water. A typical case is
calcium tartrate, which can be obtained by reaction of tartaric acid and
calcium chloride. Make a gel of silica by mixing aliquots of tartaric acid 1M
solution with sodium silicate solution (density = 1.06 g/cc) under continuous
stirring. Pour the solution in the GCB and introduce the guide. Wait for one
day until the silica gel is set. Then pour on top of the gel layer a solution
of calcium chloride (1M).
To harvest the crystals, pull out the guide and the
gel will be removed.
A.2. Want to make beautiful Liesegang’s
rings? It’s easy with the GCB. Prepare a 0.05 M solution of potassium iodide
gelled with agarose at 1% w/v concentration.
While the solution is still hot, pour it into the GCB. Let it cool to
room temperature and wait for 20 minutes. While waiting, prepare a lead nitrate
Pb(NO3)2 1M solution. Pour onto the already gelled KI
solution and close the box. That’s all.
Note that the concentration of lead
nitrate was selected to be much higher than the concentration of potassium
iodide and therefore lead molecules will invade the gelled iodide solution. As
soon as they meet, an amorphous phase of PbI2 forms. The
precipitation process continues producing intermittent precipitation. As new
bands form they are made by fewer crystals of larger size. This is exactly the foundation
of the counter-diffusion technique for protein crystallization. The difference
is the type of reaction used to trigger the precipitation. Here a precipitation
reaction occurs in which both components (lead and iodide) enter in the
crystals. Unlike this, in the case of protein crystallization, precipitation is
triggered by solubility reduction. Therefore only the protein concentration
decays noticeably.
A.3. Crystallization of a protein by
solubility reduction driven by ionic strength. This method is designed to
prepare many large high-quality crystals for special purposes but it consumes a
larger amount of protein than usual cases. Here I describe the recipe to make
tetragonal crystals of the model protein lysozyme.
Make a sol of agarose
at 1% by heating the mixture under continuous stirring above the gelling point.
Prepare a solution of lysozyme at 40 mg/ml. Let the agarose cool down to a
temperature of about 35ºC. Keep the agarose sol at this temperature. Mix while
stirring one part of agarose sol with four parts of protein solution. Pour the
mixture into the GCB and introduce the guide. Finally, pour in a solution of
NaCl at 20% w/v.
B) Growth of protein crystals inside capillaries with
un-gelled precipitating agent by the counter-diffusion technique.
The growth of biological macromolecules inside
capillaries has many advantages. Among them are: a) the scanning a large number
of crystallization conditions in a single experiment; b) the avoiding of
manipulation of crystals after they are obtained: you can use for X-ray
diffraction the same capillaries where the crystals were grown; c) to grow the
crystals under diffusion controlled mass transport.
To perform the experiment with the GCB is
very easy. The first thing to do is to prepare the agarose gel. Agarose is a
polysaccharide that does not interact chemically with most proteins (note that
in some cases you can also use silica gel). To make it, mix the appropriate
volume of buffer solution with agarose powder, under continuous stirring, for
an final agarose concentration of 1.5 % w/v. Heat the mixture to boiling in
order to break the cross-links of the agarose fibers. Note that the agarose
solution becomes transparent. Maintain the solution boiling about two minutes
under continuous stirring. Poor the agarose sol in the GCB and let it cool at
room temperature. The cross-links of the agarose will reform and a gel will be
made.
Once the gel is set, fill the capillaries with
the protein solution. To do this, introduce one of the ends of the capillary
into the protein solution. You will see that the solution flows up by
capillarity. Once it reaches a height of five or six centimeters, remove the
capillary. You will see that the solution remains inside the capillary. Then
seal the upper end of the capillary with a small piece of plasticine or your
preferred sealing material.
The next step is to punch the capillary in the
gel layer (but be sure that the gel is set!!!). Introduce the capillary through
one of the holes in the guide of the GCB and push it into the gel about 2-3 mm
(just to maintain it straight). Finally, pour the solution of your buffered
precipitating agent onto the gel layer and place the cover on the GCB. Pour a
volume equal to the volume of the gel layer. Note that you can use up to six
capillaries per GCB. This is termed the gel acupuncture method.
C) Growth of protein crystals inside capillaries with
gelled precipitating agent by the counter-diffusion technique.
The procedure is basically the same
as above. However, here you can takes advantage of the possibility to
gel some buffers and common precipitating agents. For instance, ammonium sulfate, polyethylene
glycol, sodium chloride and others can be incorporated into agarose gels as
well as most classical buffers.
To do this, just mix the agarose with your buffered starting precipitating agent solution. In other words, replace the water used to mix the agarose in the above protocol with your precipitating agent solution. Once the gel is set, just punch your capillary containing the protein solution and wait. Note that you can use the protein at any pH value at which it is stable. Because of the large volume of the gelled solution compared with the volume of the protein solution in the capillary, the pH value of the protein solution will change as the buffered precipitating agent moves into the capillary.
D) Growth of protein crystals inside capillaries by the
batch method.
You can also use the GCB to grow crystals by the microbatch method. Obviously you lose all the advantages of the counter-diffusion techniques for screening of crystallization conditions. However, if you want to try this traditional method and to grow crystals ready for X-ray diffraction inside the capillaries, you can use the GCB to hold the capillaries, to have an easy optical observation and to transport them.
To do this, prepare your buffered precipitating agent solution and mix it with agarose at 0.1 %. Boil it for 1 minute and then cool it to 35 ºC. Maintain the sol at this temperature. Mix the appropriate volume of the protein solution with the appropriate volumes of the sol. In other words, proceed as to prepare a drop for microbatch. Then, suck the drop into the capillary by capillarity. Seal both ends of the capillary and hold them in the GCB.
How much protein?
The answer to this important question depends obviously on the
diameter of the capillary chosen. We recommend filling the capillaries up to a
length of 50 or 60 mm in order to have a wide screening of the phase diagram.
The plot on the left shows the amount of protein solution you need for the
experiment as a function of the inner diameter of the capillary. The values are
listed in the table below:
|
diameter |
Length = 50 mm |
Length = 60 mm |
|
0.1 mm |
0,39 ml |
0,47 ml |
|
0.2 mm |
1,57 ml |
1,88 ml |
|
0.3 mm |
3,53 ml |
4,24 ml |
|
0.4 mm |
6,28 ml |
7,53 ml |
|
0.5 mm |
9,81 ml |
11,78 ml |
Note that you can perform screening with 0.1 mm
capillary using a protein solution volume of less than 500
nanoliters per experiment !!!.
How counter-diffusion techniques work?
The precipitation of the protein occurs because its
solubility varies with, for instance, the ionic strength, i.e., with the
concentration of salt (see the ideal phase diagram in the Figure on the left).
Let the starting protein concentration in the capillary be C0p and
the concentration of the salt be C0s. When these solutions come into
contact near one of the ends of the capillary, the system moves towards the
point C0 in the phase diagram. Note that the supersaturation is very
high. Therefore, the first precipitate will be an amorphous or ill-defined
crystalline phase forming in the lower end of the capillary. Its formation
depletes the concentration of protein in the neighboring zones. As the salt
continues to diffuse up in the capillary, a new precipitation takes place, this
time at lower supersaturation producing microcrystals (location C1
in the phase diagram). Iteration of this process provokes precipitation at
lower supersaturation as the precipitation front moves far from the gel towards
the upper part of the capillary (C2, C3, ….. Ce).
This yields precipitation zones of fewer crystals of larger size and higher
quality. Unlike the classical drop and batch methods, the counter-diffusion
technique explores a large number of crystallization conditions in one single
experiment.
Note that the crystallization pathway evolves
always towards equilibrium and therefore the experiment self-searches the
optimal crystallization scenario. The experiment is therefore equivalent to
making a large number of hanging-drop or batch experiments across the phase
diagram. Counter-diffusion techniques make such a search automatically in one
single experiment. And in addition you do not need to check the crystals in
different drops to look for those of higher quality. In the counter-diffusion
experiment the best crystals are always those formed in the upper part of the
capillary. If an X-ray capillary is used, you can collect diffraction data
directly without touching the crystals.
Do not worry about the starting conditions.
Just select a typical concentration for protein solutions to be crystallized,
for instance 10 to 20 mg/ml. Then use a very high concentration of
precipitating agent to trigger the protein precipitation at high
supersaturation as soon as the precipitating agent meets the protein solution.
As discussed above the system will evolve itself towards better crystallization
conditions.
It is important to note that in any counter-diffusion experiment
there are three main parameters (See Figure on the right):
a) the length of the reservoir where the precipitation will occur (red). This reservoir contains the compound to be crystallized (for instance a protein) or, in the case of crystallization by chemical reaction, the reactant of smaller diffusivity or at lower concentration.
b) the length of the physical buffer (yelow). This is a gel layer which does not interact chemically in the crystallization process. Its function is to slow down the mixing of the solutions. Note that this buffer layer can exist or not. For instance in the gel acupuncture method, the length of the buffer is double the depth of penetration of the capillary in the gel (before reaching the protein, the precipitating agent must travel the penetration depth down –outside the capillary- and up –inside the capillary-. However, in the case of the method using gelled precipitating agent, there is no such a buffer layer. The precipitant is in direct contact with the protein.
c) the relative values of the volume (in blue) of the precipitating agent (for the case of protein) or the component with lower concentration or smaller diffusivity (for the case of chemical reaction) with respect to the volumes in red and yellow.
It is critical to consider the values of these parameters and the initial concentration of the reactants to understand the evolution of the experiment. Depending on the configuration used, the system will evolve differently. With the configuration of the gel acupuncture method, the salt on top of the gel diffuses down through it and after a given time, reaches the punched end of the capillary. Then, it continues diffusing up through the capillary filled with the protein solution, triggering its precipitation. Note that with this configuration, the concentration of the precipitation agent at the end of the experiment will be the initial concentration of precipitating agent divided by:
Volume of the precipitating agent + Volume of the gel layer + Volume of the capillary (negligible)
--------------------------------------------------------------------------------------------------------------------
Volume of the precipitating agent
For instance, if we use a volume of precipitating agent (in blue) equal to the volume of the gel layer (yellow), the final concentration of precipitating agent in the capillary will be half of the initial concentration. Selecting the values of these volumes, we can tune how wide the screening of the phase diagram will be.