Preparing for IEF

The components of the Immobiline^® DryStrip Kit include a tray and electrode holder, anode and cathode electrodes, an Immobiline^® DryStrip aligner, a sample cup bar, and sample cups.

Procedures A and B below should be completed before the Immobiline^® DryStrip gels are removed from the Immobiline^® DryStrip IPGbox.

A. Prepare the Immobiline^® DryStrip Kit

1. Clean all components of the Immobiline^® DryStrip Kit

The Immobiline^® DryStrip tray, Immobiline^® DryStrip aligner, electrodes, sample cup bar, and sample cups must be clean and ready for use. Clean with detergent, rinse thoroughly with distilled water, and allow to dry.

2. Confirm electrical connections on MultiPhor™ II Electrophoresis System

Check that the red bridging cable in the MultiPhor™ II unit is connected (seated under the cooling plate).

3. Establish cooling

Set the temperature on MultiTemp III Thermostatic Circulator to 20 °C. Position the cooling plate on the MultiPhor™ II unit and ensure that the surface is level.

4. Position the Immobiline^® DryStrip tray

Pipette approximately 3–4 ml of Immobiline^® DryStrip Cover Fluid onto the cooling plate. Position the Immobiline^® DryStrip tray on the cooling plate so the red (anodic) electrode connection of the tray is positioned at the top of the plate near the cooling tubes. Remove any large bubbles between the tray and the cooling plate; small bubbles can be ignored. Immobiline^® DryStrip Cover Fluid serves as an electrical insulating fluid to ensure good thermal contact between the cooling plate and the tray. Connect the red and black electrode leads on the tray to the MultiPhor™ II unit.

5. Position the Immobiline^® DryStrip aligner

Pour approximately 10 ml of Immobiline^® DryStrip Cover Fluid into the Immobiline^® DryStrip tray. Place the Immobiline^® DryStrip aligner, 12-groove side up, into the tray on top of the Immobiline^® DryStrip Cover Fluid. The presence of air bubbles between the strip positions under the aligner will not affect the experiment. For easier visualization of the grooves in the aligner, avoid getting Immobiline^® DryStrip Cover Fluid on top of the aligner.

B. Prepare electrode strips

1. Cut electrode strips to size

Cut two IEF electrode strips to lengths of 110 mm each.

2. Soak electrode strips with distilled water

Place the electrode strips on a clean, flat surface such as a glass plate. Soak each electrode strip with

0.5 ml distilled water. Blot with filter paper to remove excess water.

Electrode strips must be damp, not wet. Excess water may cause streaking.

C. IEF with rehydration loading

1. Remove the rehydrated Immobiline^® DryStrip gel from the Immobiline^® DryStrip IPGbox

To remove an Immobiline^® DryStrip gel from the Immobiline^® DryStrip IPGbox, slide the tip of a pair of forceps along the sloped end of the channel and into the slight depression under the Immobiline^® DryStrip gel. Grasp the end of the strip with the forceps and lift the strip out of the tray.

2. Position the Immobiline^® DryStrip gel in the Immobiline^® DryStrip aligner

Immediately transfer the rehydrated Immobiline^® DryStrip gels (gel side up) to adjacent grooves of the aligner in the Immobiline^® DryStrip tray (Fig 1). Place the strips with the acidic ends at the top of the tray near the red electrode (anode). The other ends should be at the bottom of the tray near the black electrode (cathode). Align the Immobiline^® DryStrip gels so the anodic gel edges are lined up.

3. Attach the electrode strips

Place the moistened electrode strips laterally across the cathodic and anodic ends of the aligned Immobiline^® DryStrip gels. The electrode strips must at least be in partial contact with the gel surface of each Immobiline^® DryStrip gel.

4. Position the electrodes

Each electrode has a side marked red (anode) or black (cathode). Align each electrode over an electrode strip, ensuring the marked side corresponds to the side of the tray giving electrical contact. When the electrodes are properly aligned, press them down to contact the electrode strips. Check that the Immobiline^® DryStrip gels are still aligned in their grooves (Fig 2).

5. Overlay the Immobiline^® DryStrip gel with Immobiline^® DryStrip Cover Fluid

Overlay each Immobiline^® DryStrip gel with 3 ml of Immobiline^® DryStrip Cover Fluid to minimize evaporation and urea crystallization.

Figure 1. Positioning Immobiline^® DryStrip gels in the Immobiline^® DryStrip aligner.

Figure 2. Alignment of electrodes over Immobiline^® DryStrip gels.