1. Clean and dry the IPGphor 3 bed before placing the Manifold tray on the unit. Position the Manifold on the IPGphor 3 platform. The small T-shaped protrusion fits into a cutout section of the IPGphor bed near the lid hinge (Figure 1). Ensure that the Manifold is level by placing the round spirit level on the center of the Manifold tray after it is placed on the Ettan™ IPGphor 3 unit. Adjust leveling feet if necessary.
Important! Before proceeding, make sure the Ettan™ IPGphor 3 unit is placed on a level surface.
Important! If using the original Ettan™ IPGphor, ensure that the three foam pads have been removed from the lid of the unit. (This step is not necessary if using Ettan™ IPGphor 3.)
2. Measure out 108 ml of Immobiline® DryStrip Cover Fluid (even if fewer than 12 strips will be loaded into the Manifold). Add the cover fluid evenly between the 12 Manifold channels. Transfer the strips to the Ettan™ IPGphor Manifold. Place the strips under the cover fluid, gel side up in the tray with the anodic (+) end of the IPG strip oriented toward the anodic side of the instrument. Position the strip to rest on the appropriate mark etched into the bottom of the Manifold channel (the end of the gel, not the end of the plastic, should align with the etched mark). Center the strip down the length of the Manifold channel. Protrusions along the sides guide the strip approximately straight, although some manual adjustment of the strip may be necessary (Figure 2).
Note: If cathodic cup loading is going to be used, the strips should be placed such that the anodic end of the strips is 3–4 cm beyond the etched placement mark.
3. If performing cup loading, place a strip of cups in the appropriate position (Figure 3), for example ~1 cm from the end of the gel portion of the IPG strip. Do NOT place the cup with the feet over a center protrusion. Push the cups into the channels with gloved fingers, starting at one end and working toward the other. Align the insertion tool over the cups and push down to ensure that the feet of the cups are properly seated at the bottom of the channel (wiggle the tool gently while pushing down in order to ensure that the cups are seated as far down as they will go). Take care not to move the cups while removing the insertion tool. Ensure that the cups are filled with cover fluid.
If desired, test for leakage by adding some colored sample buffer (without sample). If no leaks are detected, pipette the colored liquid back out again.
Cups must not straddle the centering protrusions on the bottom of the channels.
4. Count out the appropriate number of precut paper wicks. Two wicks per strip are required. Separate the wicks from each other. Add 150 µL of distilled water to each wick. Place the wicks on the IPG strips such that one end of the wick overlaps the end of the gel on the IPG strip (Figure 4). For gradients with pH above 9, add 150 µL DeStreak rehydration solution to the cathodic wick. The electrode must contact the wick. With the electrode cams in the open position, place the electrode assembly on top of all the wicks. Swivel the cams into the closed position under the external lip of the tray. The electrodes should not be moved while the cams are in the closed position (Figure 5).
5. Briefly centrifuge the protein sample (e.g. at top speed in a microcentrifuge) prior to loading to remove insoluble material and particulate matter. These materials could impede sample entry and result in vertical streaks in the second-dimension gel. Load samples into the sample cups. A maximum of 150 µL of sample may be placed in these cups. Check to make sure that there is cover fluid over the samples. When the cups are initially placed on the Manifold, cover fluid will flow into the cups as they are seated. When sample is introduced into the cups, the sample will sink to the bottom of the cup and contact the IPG strip.
Note: For basic IPG strips, superior focusing patterns are generally obtained when the sample cup is placed as close to the anodic (+) electrode as possible.
6. Close the Ettan™ IPGphor 3 lid. Program the Ettan™ IPGphor 3 with the desired run parameters. Ramping the voltage slowly while the sample is entering the IPG strip will improve results. See section 2.8 for further discussion on this topic. Optimal ramp, voltages and times, or Vhr (volt-hours) totals must be determined empirically for each sample type. Focusing after sample cup application frequently requires fewer Vhr than in-gel sample rehydration loading methods, particularly on basic pH-range strips.
Figure 1. Manifold placement on Ettan™ IPGphor 3
Figure 2. Placement of IPG strips in Manifold channels. Note: If cathodic cup loading is going to be used, the strips should be placed such that the anodic end of the strips is 3–4 cm beyond the etched placement mark.
Figure 3.Sample cup positioning details Note: Cups must not straddle the centering protrusions on the bottom of the channels.
Figure 4.Correct placement of paper wicks
Figure 5.Placement of electrode on paper wicks. Cams are in the open position.