Crystallization screening directly in electrophoresis gels

Journal of Crystal Growth 232 (2001) 596–602
Crystallization screening directly in electrophoresis gels
J.M. Garcı́a-Ruiza,*, A. Hernández-Hernándeza, J. López-Jaramilloa, B. Thomasb
Laboratorio de Estudios Cristalográficos, Institut Andaluz de Ciencias de la Tierra CSIC-Universidad de Granada. Av. Fuentenueva s/n,
18002 Granada, Spain
Center for Microgravity and Materials Research, Research Institute, Room D29. University of Alabama, Huntsville, AL 35806, USA
Screening of crystallization conditions can be directly performed within electrophoretic gels run during the
purification steps. The technique is demonstrated for different methods of electrophoretic separations and for two
different gels, agarose and polyacrylamide. Two different techniques are proposed to perform the screening: direct
diffusive mixing in small gel slices and diffusion technique in gel bands. # 2001 Elsevier Science B.V. All rights reserved.
Keywords: A1. Biocrystallization; A1. Gel electrophoresis; A2. Growth from solutions; B1. Biological macro-molecules; B1. Proteins
1. Introduction
Electrophoresis is the method currently used by
biochemists and molecular biologists to separate
biological macromolecules (nucleic acids and
proteins) on the basis of size, electric charge, and
other physical properties [1]. In practical terms, the
molecules under the effect of an electrical current
are forced to migrate across a span of gel that
works as a molecular sieve. Activated electrodes at
either end of the gel provide the driving force. The
rate of migration through the electric field depends
on the strength of the field, size and shape of the
molecules, relative hydrophobicity of the samples,
and on the ionic strength and temperature of the
buffer in which the molecules are moving. As a
result, the separated macromolecules form a
number of bands perpendicular to the direction
of the electric field [1]. Isoelectric focusing is
*Corresponding author. Tel.: +34-958-243360; fax: +34958-243384.
E-mail address: [email protected] (J.M. Garcı́a-Ruiz).
another electrophoretic technique that works by
the formation of a pH gradient across the gel by
carrier ampholytes. The ampholytes are mixed in
the supplied gel. For instance, in the PhastSystem,
the ampholytes are first placed in the electric field
to create the pH gradient and then the samples are
applied. In the electric field the proteins migrate to
their isoelectric points where the overall charge on
the protein is neutral. The PhastGels are available
without ampholytes so that the range needed can
be set by adding the appropriate ampholytes to the
gel surface. On other hand, it is also known that
gels can be used as growth media to crystallize
inorganic and organic compounds [2–5]. The ‘‘gel
technique’’ is known to work for thermal gels (for
instance, agarose) as well as for chemical gels
(silica or polyacrylamide gels).
Because native gel electrophoresis will separate
protein impurities based on charge and size, and
isoelectric focusing will separate impurities with
high resolution based on charge differences, we
have explored the possibility of utilizing gels
from native electrophoresis to screen pH and
0022-0248/01/$ - see front matter # 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 2 2 - 0 2 4 8 ( 0 1 ) 0 1 0 8 3 - 1
J.M. Garcı´a-Ruiz et al. / Journal of Crystal Growth 232 (2001) 596–602
precipitating agents for crystallizing abilities. It
would save time and protein in the route to
obtain crystals. Thus, in a quick and simple way,
the protein of interest is purified to remove
impurities, which may inhibit crystallization. The
purified protein in the gel is then subjected to
crystallization by counter diffusion or other
techniques. In some cases the need for time
consuming chromatographic purification methods
may be circumvented. The method is demonstrated to work for two different types of gels,
two different crystallization techniques, and two
different proteins.
2. Experimental procedure
Polyacrylamide and agarose gels were tested
because they are commonly used for electrophoresis. Hen egg white lysozyme (pI=11.4) and horse
spleen ferritin (pI=4.3) were chosen as test
proteins because of these pI values. Two different
crystallization methods were designed and are
described below.
Native horizontal electrophoresis in agarose gels
was carried out with a submarine electrophoresis
system purchased from BIO-RAD. The gel was
made of Agarose l (code 17-0424-02) from
Pharmacia Biotech, at concentration 0.5 and 1%
w/v. Ferritin in the gel was buffered to pH 7.5 with
200 mM Tris-HCl solution. Six 20 ml lanes of
25 mg/ml horse spleen ferritin (Fluka-4503 lot
125H70402) were loaded. For lysozyme (Fluka62971 (lot: 331429/1595), five or six 40 ml lanes at
concentration ranging from 100 to 200 mg/ml were
tested. The applied voltage was 100 V for 60 min
for ferritin and 100 V for 120 min for lysozyme.
Native electrophoresis in 7.5% polyacrylamide
gels was carried out with a BIO-RAD MiniProtean II. The gel was buffered to pH 8.3 with
250 mM Tris-glycine solution. Ten 10 ml lanes of
12.5 mg/ml horse spleen ferritin were performed. A
voltage of 150 V was applied for 75 min.
For isoelectric focusing, the procedure was also
a classical one. A single lane sample applicator
(normally used for 2D gels) was used to apply
100 mg/ml ferritin (Sigma Chemical Co. St. Louis,
MO, USA) to an Amersham Pharmacia Biotech
(Piscataway, NJ, USA) 3-9 pI isoelectric focusing
PhastGel. Since the ampholytes act as buffers, the
precipitating agent was dissolved in a concentrated
buffer (0.2 M) to adjust the pH to 5.0.
Two different screening methods are used after
separation is achieved:
(a) Gel slices: It consists of soaking small pieces
(2 2 mm) of gel containing the protein bands
into the precipitating agent solution. This
screening method is similar to a batch method.
However, unlike batch where precipitating
agent and protein are directly mixed, this
method allows the precipitant to diffuse into
the gel slice. Because the gel layer is thin,
diffusive mixing occurs quickly. After mixing
occurs, the band of gel containing the protein
is cut in small pieces. To precipitate the
protein, various concentrations of precipitating agents previously buffered at the desired
pH are poured onto each slice of gel.
Cadmium sulfate at concentration ranging
from 2 to 10% (w/v) buffered at pH=5.3 with
200 mM sodium acetate were used for ferritin
experiments. Sodium chloride at concentrations of 13–30% (w/v) buffered to pH=4.5
with 50 mM sodium acetate were used for
lysozyme experiments.
(b) Whole band: This screen is actually a counterdiffusion experiment. It consists of using the
whole band of gel containing the protein and
soaking one of its sides perpendicular to the
protein bands in the solution of the precipitating agent. The precipitating agent diffuses into
the gel producing a gradient from high to low
supersaturation. Because of their flexible
plastic backing, PhastGels can be inserted
into a solution of precipitating agent to a
depth of 0.5 cm. For native electrophoresis,
the whole band of gel containing the protein is
oriented in the precipitating agent so that the
protein band is perpendicular to the precipitating agent, allowing it to diffuse along the
protein band. Cadmium sulfate (10% w/v in
200 mM sodium acetate at pH=5.0) was used
to precipitate ferritin while 30% (w/v) NaCl in
50 mM sodium acetate at pH=4.5 was used to
precipitate lysozyme.
J.M. Garcı´a-Ruiz et al. / Journal of Crystal Growth 232 (2001) 596–602
3. Results and discussion
The use of native horizontal electrophoresis is
demonstrated using 100 mg/ml lysozyme and 1%
agarose. Fig. 1 shows precipitates obtained at
different concentrations of NaCl using gel slices.
Crystals appear at 15% NaCl, while a higher NaCl
concentration yields spherulites and sheaf-ofwheat morphologies characteristic of high supersaturation.
The use of the whole band with counterdiffusion method is illustrated by a crystallization
screening inside electrophoretic gels of agarose
Fig. 1. Lysozyme precipitates obtained at different concentration of NaCl. From top to bottom, 30, 20 and 15% (w/v).
containing isolated bands of ferritin (Fig. 2). The
precipitating agent (CdSO4) diffuses from the
bottom. The screening is performed therefore from
high supersaturation (lower part) to low supersaturation (upper part). Observations confirmed
what is expected from the counter-diffusion
technique. Thus, in the lower part fractal dendrites
are observed, followed by a region of spherulites,
later a region of dendritic crystals and finally a
region where faceted octahedral crystals form.
This type of screening permits scanning of a wide
range of concentrations for a given precipitating
agent and a given pH value. Note for instance in
the above results that crystal dendrites appear in a
wide range of crystallization conditions, while the
range for octahedral crystals is very narrow. This
is in agreement with the precipitation behavior of
ferritin in presence of CdSO4 concentration
obtained in a separate work [6].
This precipitation behavior of ferritin was
confirmed by the technique of direct mixing of
precipitating agent with gel slices containing the
protein. Fig. 3 shows the results obtained as a
function of the CdSO4 concentration. Spherulites
are obtained at a concentration of 10% (w/v).
Dendritic forms occur at 4 at 8% CdSO4 while
crystals become faceted at 4%. Octahedral crystals
appear at 2% CdSO4.
The technique also works for those cases in
which the protein is no longer pure because of
polymerization, denaturation or just contamination with foreign compounds. To illustrate this, we
present the results obtained by using polyacrylamide electrophoretic gels. As shown in Fig. 4,
three protein bands were separated. The broad and
denser one corresponds to the monomeric form of
ferritin, as checked by light scattering in a separate
experiment, while the narrow and lighter ones
correspond to the dimer and trimer. Gel slices were
cut from the three bands and drops of CdSO4 at
different concentrations were poured on them.
After 24 h, faceted crystals of ferritin (Fig. 4)
appear in the gel slices soaked in 10 and 8%
CdSO4 and they appear only on the gel slices of the
band containing monomeric ferritin. After one
month, crystals did not appear in the gel slices
containing either the dimeric or the trimeric
ferritin forms. Note also that the shift of the
J.M. Garcı´a-Ruiz et al. / Journal of Crystal Growth 232 (2001) 596–602
Fig. 2. Counter-diffusion screening performed within a 1% agarose electrophoretic gel containing a band of monomeric ferritin.
precipitation behavior to higher precipitating
agent concentration is due to the lower concentration of protein used in the electrophoretic run.
These results agree with those described by
Thomas et al. [7] for the iron free ferritin
The pore size of agarose gels is very large for
most of the proteins and they are normally used
J.M. Garcı´a-Ruiz et al. / Journal of Crystal Growth 232 (2001) 596–602
Fig. 3. Direct mixing screening using slices of a 1% agarose electrophoretic gel containing monomeric ferritin. The CdSO4
concentration (%w/v) varies as labeled in the figure.
Fig. 4. Above: a native PAGE of horse spleen ferritin. From
upper to lower, the protein bands correspond to the trimeric,
dimeric and monomeric forms. Below: examples of ferritin
crystals obtained from slices of gels containing the monomeric
for electrophoresis of nucleic acids. Therefore,
when agarose gels at a concentration of 1% are
used to electrophoretically separate a protein like
lysozyme (molecular weight of 14 kDa), 2D screening takes place when the whole band is screened
with the counter-diffusion arrangement. In the
absence of an electric field, when the counterdiffusion screening is set, the precipitant diffuses
across the band and, concurrently, the protein also
diffuses sideways broadening the band. The
combination of both diffusion trends results in a
two-dimensional V-shaped pattern which results in
a range of protein concentrations as shown in
Fig. 5. Thus, protein concentration can also be
We obviously use chemical agents known to
precipitate the tested protein in this practical
demonstration of the technique. In a test for a
new protein, one should use the whole set of the
preferred screening kit in the gel slice technique.
About fifty 1.5 1.5 mm2 gel slices can be
obtained from a 8 cm long and 0.3 cm wide band.
Therefore, 50 different chemical variations to
precipitate the protein can be tested. We recommend to use this technique to screen pH and type
of precipitating agent. All the precipitating agents
currently used in crystallization screening should
be compatible with the gel slice technique because,
J.M. Garcı´a-Ruiz et al. / Journal of Crystal Growth 232 (2001) 596–602
Fig. 5. Screening pattern obtained when the pore size of the gel (0.5% agarose gel) is much larger than the protein size (HEW
lysozyme). Precipitating agent is 30% w/v NaCl.
even if these chemicals cannot diffuse into the gel,
the protein diffuses from the gel into the precipitant solution.
the protein is crystallized just after its separation. In short, the corollary of this work is:
do not discard your gels after electrophoresis,
use them to search for potential crystallization
4. Conclusion
We demonstrate that different electrophoresis
gels can be directly used for screening of crystallization conditions after being used to analyze
the protein during the purification steps. The
use of this method drastically reduces the
waste of protein in further screening as it uses
a material that is often discarded after use in
the previous and unavoidable process of purification. In addition, for those proteins that have
a strong tendency to denature or to aggregate,
this method represents an advantage because
This research has been supported by the Spanish
Ministerio de Educación y Ciencia, Consejo
Superior de Investigaciones Cientificas and European Space Agency. J.M. Garcı́a-Ruiz also
acknowledge the University Space and Research
Association for a stay at the Marshall Space Flight
Center. The authors acknowledge the improvement of the clarity of the manuscript by an
anonymous referee.
J.M. Garcı´a-Ruiz et al. / Journal of Crystal Growth 232 (2001) 596–602
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