Separation of proteins using supramolecular gel electrophoresis

Article abstract of DOI:10.1039/c1cc13826j

An amphiphilic low-molecular-weight hydrogelator 1 was synthesized. A tris-glycine-SDS solution gel of 1 was applied for electrophoresis to separate proteins. Centrifugation of a mixture of protein and a hydrogel of 1 enabled the recovery of protein. Various combinations of proteins were applied for supramolecular gel electrophoresis (SUGE), and remarkably poor mobility for small proteins (<45 kDa) was found.

Full text of DOI:10.1039/c1cc13826j

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COMMUNICATION
Separation of proteins using supramolecular gel electrophoresisw
a
a
Sachiyo Yamamichi,z Yuki Jinno,z Nana Haraya,a Takanori Oyoshi,a Hideyuki Tomitori,b
Keiko Kashiwagib and Masamichi Yamanaka*a
Received 27th June 2011, Accepted 8th August 2011
DOI: 10.1039/c1cc13826j
An amphiphilic low-molecular-weight hydrogelator
1
was
indicates that the fine-tuning of gels enables the development
of specialized electrophoresis. The structural divergence of
supramolecular hydrogels favors the creation of electrophoretic
techniques according to a specific purpose. In this manuscript,
we report the synthesis of an amphiphilic low molecular
weight (LMW) hydrogelator and its application to the electro-
phoresis of proteins. Efficient recovery of proteins and drama-
tically poor mobility of small proteins were observed using this
supramolecular gel electrophoresis (SUGE) approach.
synthesized. A tris-glycine-SDS solution gel of 1 was applied
for electrophoresis to separate proteins. Centrifugation of a
mixture of protein and a hydrogel of 1 enabled the recovery of
protein. Various combinations of proteins were applied for
supramolecular gel electrophoresis (SUGE), and remarkably
poor mobility for small proteins (o45 kDa) was found.
Self-assembly of preorganized small molecules is a convenient
approach to nanoscale architecture. A supramolecular gel
composed of fibrous aggregates interlinked noncovalently is
an attractive target in this area.1 Most importantly, research
on supramolecular hydrogels has been expanding in the last
decade, because of their potential for biological applicability.2
Great progress has been attained especially in the fields of
tissue engineering or chemosensors.3 In contrast, electrophoresis
using supramolecular hydrogels remains as an undeveloped
area of research. Gel electrophoresis is one of the most funda-
mental and frequently used techniques in the analysis of
diverse biomolecules.4 Polyacrylamide gel electrophoresis
(PAGE), which was first reported in the middle of the last
century, and sodium dodecyl sulfate PAGE (SDS-PAGE) play
a central role in the analysis of proteins.5 The application of
supramolecular hydrogels to protein electrophoresis has
potential advantages compared with polyacrylamide gels. One
of them is the efficient recovery of protein samples. Polyacryla-
mide gels show strong affinity for proteins; thus, the recovery
of proteins after PAGE is laborious.6 The resolvable nature of
supramolecular hydrogels is an advantage regarding the recovery
of proteins, as the resolved gel would show lower affinity for
proteins compared with the original gel. The other advantage
is the potential for a novel separation manner depending on
the structural nature of supramolecular hydrogels. A Phos-tag
copolymerized polyacrylamide gel reported by Koike, Kinoshita,
and co-workers exhibited high affinity for phosphorylated
proteins, and attained unique electrophoresis.7 This result
C3-symmetric tris-urea LMW organogelators have been
developed in our laboratory.8 Peripheral modification of the
organogelator with hydrophilic groups afforded an amphiphilic
LMW hydrogelator.9 The amphiphile 1 was designed and
synthesized from commercial pentaacetyl a-D-glucoside using
eight-step reactions (Fig. 1). A mixture of 1 and Tris-Glycine-SDS
(TGS) solution (Tris: 25 mM; Glycine: 192 mM; SDS: 0.1%)
yielded a hydrogel after brief heating; the minimum gelation
concentration of 1 was estimated at 1.5 wt% (Fig. 2 inset). The
hydrogel was stable at ambient temperature for a long period,
and kept a high transparency, even at 5.0 wt%. The thermal
10
stability (Tgel
)
of the hydrogel was increased by increasing
the concentration of 1 as a typical characteristic of the
supramolecular gel (Tgel; 46 1C for 1.5 wt%, 92 1C for
2.0 wt%, 4100 1C for 3.0 wt%). Narrow fibers (average
diameter = 70 nm) were observed in the scanning electron
microscopy (SEM) images of the xerogel (Fig. 2).
a Department of Chemistry, Faculty of Science, Shizuoka University,
836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
E-mail: smyaman@ipc.shizuoka.ac.jp; Fax: +81 54 237 3384;
Tel: +81 54 238 4936
b Faculty of Pharmacy, Chiba Institute of Science, 15-8 Shiomi-cho,
Choshi, Chiba 288-0025, Japan
w Electronic supplementary information (ESI) available: Synthetic
procedures, spectral data, gelation experiments, procedure of electro-
phoresis, and Fig. S1–S4. See DOI: 10.1039/c1cc13826j
z These authors contributed equally to this work.
Fig. 1 Chemical structure of LMW hydrogelator 1.
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Fig. 2 Photograph and SEM image of TGS solution gel of 1.
The aptitude of the TGS solution gel of 1 for electrophoresis
aimed at separating proteins was tested. A capillary (f = 1.7 mm)
was filled with the TGS solution gel of 1, and a color dye
conjugated protein marker (DynaMarkers Protein Multi-
Color III) was applied on one side. The capillary was applied
to electrophoresis using a submarine electrophoresis system.
The TGS solution gel of 1 held out against electrophoresis
conditions. Six faded bands were observed after the electro-
phoresis (100 V, 40 min) using a 2.0 wt% TGS solution gel of 1;
however, a 12 wt% polyacrylamide gel filled capillary sepa-
rated all eight proteins after submarine electrophoresis (135 V,
40 min) (Fig. S1, ESIw). The gel of 1 was too fragile to handle
outside the capillary; therefore, it was difficult to analyze its
exact separation capacity in detail. Accordingly, a mixed TGS
solution gel consisting of 1 (2.0 wt%) and agarose (2.0 wt%)
[1-AG gel] was used in subsequent electrophoretic experiments.
The gel was sufficiently hard to handle outside the capillary,
even after electrophoresis, and the agarose gel itself had no
separation ability for proteins, at least in the range from 16 to
229 kDa.
Fig. 3 Schematic representation of SDS-SUGE and following
SDS-PAGE analysis.
A mixture of b-galactosidase (116 kDa) and ovalbumin
(45 kDa) was employed for SDS-SUGE using the 1-AG gel
(100 V, 120 min.) (Fig. S2, ESIw). Smaller ovalbumin was
detected in lanes 4 to 7, and a stronger band was found in lane
5. Larger b-galactosidase was detected in lanes 6 to 8, with the
strongest band observed in lane 7. Ovalbumin was electro-
phoresed more to the anode side than b-galactosidase as well
as in SDS-PAGE, however the separation was dull (Fig. 4a).
Next, we chose a combination of ovalbumin and the much
smaller lysozyme (14.4 kDa). SDS-SUGE was performed at
100 V for 170 min and the separation pattern was analyzed
using SDS-PAGE followed by CBB staining (Fig. 4b and
Fig. S2, ESIw). Ovalbumin was detected in lanes 3 and 4, whereas
the smaller lysozyme was detected in lane 5. This result means
that the smaller protein was retained closer to the cathode side
compared with the larger one, i.e., SDS-SUGE using the LMW
hydrogelator 1 yielded a separation pattern that was different
from that observed in typical SDS-PAGE. To identify the
generality of this unusual separation pattern, aprotinin (6.5 kDa)
was used in SDS-SUGE, instead of lysozyme. Electrophoresis
of ovalbumin and aprotinin was performed at 100 V for 150 min
(Fig. S2, ESIw). SDS-PAGE analysis showed that ovalbumin
was distributed in lanes 3 to 5, and the smaller aprotinin was
found in lane 7 (Fig. 4c). These results indicate that SDS-
SUGE using the LMW hydrogelator 1 exhibits remarkably
poor mobility for small proteins. Two different molecular sieve
effects should be added to this electrophoretic technique. One
is analogous to the separation mechanism of typical SDS-
PAGE. String-like denatured proteins would pass through the
three-dimensionally intertwining fibrous network of 1, and
smaller proteins would show large mobility. The other resembles
the separation mechanism of gel filtration. Isolated spaces
Isolation of proteins from the 1-AG gel was achieved using
an extremely simple centrifugation step. The 1-AG gel containing
electrophoresed proteins was centrifuged (14 100 g). The super-
natant was applied to SDS-PAGE, together with weighted
references. Quantitative evaluation of extracted proteins was
accomplished via analysis of a coomassie brilliant blue (CBB)
stained polyacrylamide gel plate using the ImageJ software.
Extraction of ovalbumin and lysozyme was attained with a
yield of up to 27 and 43%, respectively. Other proteins were
also recovered using this procedure, and their yields were
about 20 to 40%. In contrast, this procedure was not effective
for the polyacrylamide gel, and no proteins were detected in
the extracts.
A typical procedure of SDS-SUGE using the 1-AG gel is
shown in Fig. 3. A capillary (f = 2 mm, length = 120 mm) was
filled with the 1-AG gel (80 mm), a mixture of pre-denatured
proteins was adsorbed onto an end of the gel, and both ends
of the capillary were filled with an agarose gel (2.0 wt%).
The capillary was sunk in TGS solution in the submarine
electrophoresis system and electrophoresed using optional
voltage and time. The electrophoresed gel was taken out of
the capillary and divided into eight equal parts (numbered 1 to
8 from the anode side). Extracted solutions were analyzed
using typical SDS-PAGE and CBB staining.
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was performed at 100 V for 170 min. SDS-PAGE analysis
showed that ovalbumin was identified mainly in lanes 4 and 5,
and the smaller carbonic anhydrase was detected in lane 6
(Fig. S3, ESIw). The relatively scanty separation of ovalbumin
and carbonic anhydrase seems to support the above-mentioned
mechanism. SDS-SUGE allowed the separation of small-size
proteins. A mixture of lysozyme (14.4 kDa) and aprotinin
(6.5 kDa) was used for SDS-SUGE at 100 V for 180 min
(Fig. S2, ESIw). SDS-PAGE analysis showed that lysozyme
was mainly found in lane 5, and smaller aprotinin was retained
at the more cathodic lanes 6 and 7 (Fig. 4d). Electrophoresis
using the TGS solution gel of 1 (1.5 or 3.0 wt%) and agarose
(2.0 wt%) yielded similar separation tendencies; however, the
relative mobility changed slightly (Fig. S4, ESIw). This suggests
that the unique separation originates in the properties of the
pseudopolymer of 1 itself.
In conclusion, we have demonstrated the potential of
supramolecular hydrogels as a basis for electrophoresis. Some
advantages, such as a simple extraction procedure and unique
separation pattern, may render this technique superior to
the classical agarose or polyacrylamide gel electrophoreses.
The development of a practical procedure of supramolecular
hydrogel electrophoresis is currently being investigated in our
laboratory.
This work was partly supported by Daicel Chemical Industry
Award in Synthetic Organic Chemistry, Japan and Grant-in-aid
for the Scientific Research on Innovative Areas of Fusion
Materials: Creative Development of Materials and Exploration
of Their Function through Molecular Control (no. 2206). The
research was partially carried out using an instrument at the
Center for Instrumental Analysis of Shizuoka University.
Notes and references
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Fig. 4 SDS-PAGE analyses of SDS-SUGE (1-AG gel) separation of
(a) b-galactosidase (116 kDa) and ovalbumin (45 kDa) (12% polyacryl-
amide gel); (b) ovalbumin and lysozyme (14.4 kDa) (15% polyacrylamide
gel); (c) ovalbumin and aprotinin (6.5 kDa) (15% polyacrylamide gel);
(d) lysozyme and aprotinin (15% polyacrylamide gel).
constructed from the self-assembly of 1 may be suitable for
retaining the proper size of proteins against an electric current.
Larger proteins were chiefly influenced by the former mechanism.
SDS-SUGE of ovalbumin and carbonic anhydrase (29 kDa)
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10346 Chem. Commun., 2011, 47, 10344–10346
This journal is The Royal Society of Chemistry 2011