Increased IEF Throughput Using a Dual-Electrode Assembly

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.

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

  1. 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.
  2. 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.
  3. 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: smejkal.gary@gmail.com.

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