An isoelectric focusing (IEF)
apparatus capable of operating
at 12,000 V maximum
voltage enables isoelectric
focusing of proteins in immobilized pH
gradients (IPGs) to be completed in
less than 3 hr.1 The IEF100 (Hoefer,
Inc., Holliston, MA) is a microprocessor-controlled isoelectric focusing
apparatus in which voltage, current,
power, and temperature are controlled,
monitored, and output by a computer
interface. In its standard configuration,
the IEF100 can run up to six IPG strips
in a 26-cm plastic tray with adjustable
electrodes that can be moved along the
length of the tray to accommodate different
IPG strip lengths. When running
7-cm IPGs, twice the usual number
of strips can be run in parallel using
a dual-electrode module in which the
acidic ends of the gradients are positioned
proximally and the basic ends
of the gradients are at opposing ends of
the running tray (Figure 1).
Figure 1 - IEF100 with dual-electrode module enabling six
pairs of IPG strips to be run in parallel circuit.
A standard protein solution was prepared
from live Escherichia coli strain
OP50 cells. Approximately 100 mg
of cells was pelleted from the liquid
growth medium by centrifugation,
washed once with water, and then
resuspended in 4.5 mL of 7 M urea, 2
M thiourea, 4% 3-[(3-cholamidopropyl)
dimethyl-ammonio]-1-propanesulfonate
(CHAPS), and 5 mM tributylphosphine,
and supplemented with protease inhibitors
(Sigma-Aldrich Chemical, St. Louis,
MO). The bacteria were divided into three
PULSE tubes, each containing 1.5 mL,
and were lysed at 45,000 psi in an NEP-3229 Barocycler (Pressure Biosciences,
South Easton, MA) as previously
described.2 The proteins were alkylated
for 2 hr with 10 mM acrylamide, followed
by precipitation with 83% acetone. The
flocculants were pelleted by centrifugation
and redissolved in 4.5 mL of 7 M urea, 2
M thiourea, 4% CHAPS, 50 mM dithiothreitol
(DTT), and 0.01% 1-phenylazo-2-naphthol-6,8-disulfonic acid disodium salt
to a final protein concentration of 1 mg/mL. Sample conductivity, measured with a
Horiba B160 conductivity meter (Horiba,
Kyoto, Japan), was 88 μS/cm.
Thirty 7-cm ServaBlue IPG strips, pH 3–10
nonlinear (Serva Electrophoresis GmbH,
Heidelberg, Germany), were hydrated overnight,
each with 140 μL of the bacterial
lysate. IEF of 2 × 12 IPGs was programmed
to run in a single step for 3 hr at 12,000 V,
50 μA, and 0.6 W in two separate IEF100
units configured with dual electrodes. Current
limiting at 50 μA resulted in the formation
of a roughly linear voltage gradient
that reached 12,000 V maximum voltage in
2 hr. Temperature remained constant at 20
°C. IEF was continued at 12,000 V for an
additional 20–22 min and terminated when
12 total kVh had elapsed.
Six additional IPGs were run on the
IEF100 using the standard electrode
pair. The run was programmed as a single
step limited at 12,000 V, 25 μA, and
0.3 W. Electrophoresis reached 12,000 V
maximum voltage in 2 hr. Temperature
remained constant at 20 °C. IEF was continued
at 12,000 V for an additional 18
min and terminated when 12 total
kVh had elapsed.
Figure 2 - Identical regions enlarged from 2-D gels showing
reproducibility of duplicate IPGs run simultaneously with left (L) or
right (R) electrode pairs wired in parallel. IPGs were pH 3–10 nonlinear.
SDS-PAGE was performed on 11% polyacrylamide gels.
Corresponding IPGs run simultaneously
with the left and right electrode
pair in the dual-electrode configuration
were compared by two-dimensional
electrophoresis (2DE). For this, IPGs
were equilibrated 2 × 10 min in sodium
dodecyl sulfate (SDS) equilibration buffer
containing 3% SDS, 3 M urea, 50
mM DTT, and 0.01% m-cresolsulfonephthalein
in 375 mM Tris-HCl, pH 8.8.
Second-dimension SDS-PAGE (polyacrylamide gel electrophoresis) was
completed in 80 min in the SE640 dual
electrophoresis unit (Hoefer, Inc.).
Proteins were visualized using the colloidal
Coomassie brilliant blue (CBB)
stain described by Wijte et al.3
Duplicate IPGs run in parallel with
the dual-electrode configuration were
indistinguishable from IPGs run with
a single electrode pair. The reproducibility
of IEF100 at 12,000 V with the
dual-electrode module was further
evidenced by 2DE, as shown in Figure
2, which compares left and right electrode
pairs.
References
- Smejkal, G.B.; Bauer, D.J. 2-D in a day: a two-dimensional gel electrophoresis work flow completed in under five hours. Am.Biotechnol. Lab. 2010, 28(5), 24–7.
- Smejkal, G.B.; Robinson, M.H. et al. Increased protein yields from Escherichia coli using pressure cycling technology. J. Biomolec. Technol. 2006, 17, 159–61.
- Wijte, D.; De Jong, A. et al. ProteomIQ Blue, a potent post-stain for the visualization and subsequent mass spectrometry based identification of fluorescent stained proteins on 2D-gels. J. Proteome Res. 2006, 5, 2033–8.
The authors are with the Hubbard Center for
Genome Studies, University of New Hampshire,
Gregg Hall, 35 Colovos Rd., Durham, NH
03824, U.S.A.; tel.: 603-204-4947; e-mail: [email protected].