If the sample was not applied by means of the rehydration solution, it can be applied using the sample cups immediately prior to isoelectric focusing. When sample cups are used, the sample load limits are lower and more specific.

Protocol

1. Prepare the sample

Prepare the sample in a solution similar in composition to the rehydration solution used.

2. Determine the point of sample application

The optimal application point depends on the characteristics of the sample. When the proteins of interest have acidic pIs or when SDS has been used in sample preparation, sample application near the cathode is recommended. Anodic sample application is necessary with pH 6–11 and 6–9 gradients and preferred when pH 3–10 gradients are used. The optimal application point can vary with the nature of the sample. Empirical determination of the optimal application point is best.

3. Position the sample cup bar

Place sample cups on the sample cup bar, high enough on the bar to avoid touching the gel surface. Position the sample cup bar so the sample cups are a few millimeters away from the cathodic or anodic electrode, depending on your sample. The sample cups must face the electrode. The sample cup bar has a spacer on one side; slide the sample cup bar toward the anode/cathode until the spacer just touches the anodic/cathodic electrode.

4. Press the sample cups against the Immobiline® DryStrip gels

Move the sample cups into position, one sample cup above each Immobiline® DryStrip gel, and gently press the sample cups down to ensure good contact with each Immobiline® DryStrip gel (Figure 1). This is the most critical part of the setup. Check that strips are in their correct, straight position in the Immobiline® DryStrip aligner.

5. Apply Immobiline® DryStrip Cover Fluid

Once the sample cups are properly positioned, pour 70–80 ml Immobiline® DryStrip Cover Fluid into the tray to completely cover the Immobiline® DryStrip gels. If the Immobiline® DryStrip Cover Fluid leaks into the sample cups, remove it with a pipette, correct the leakage, and check for leakage again. Add approximately 150 ml of Immobiline® DryStrip Cover Fluid to completely cover the sample cups. The Immobiline® DryStrip gels are submerged under a layer of Immobiline® DryStrip Cover Fluid to prevent drying of the gel, precipitation of the components of the rehydration solution, and diffusion of gas into the gel.

6. Apply the sample

Apply sample (up to 100 µL per Immobiline® DryStrip gel) into the sample cups by pipetting under the surface of the Immobiline® DryStrip Cover Fluid (Figure 2). The sample should sink to the bottom of the cup. Check for leakage.

7. Start IEF

Ensure that the electrodes on the tray are connected and place the lid on the MultiPhor™ II unit. Connect the leads on the lid to the power supply. Ensure that the current check on the EPS 3501 XL Power Supply is switched off. Begin IEF.

When sample is applied via sample cups, precipitates can form at the application point and the amount of protein that can be loaded is less than if the sample was included in the rehydration solution. Protein precipitation and aggregation at the application point can sometimes be avoided by observing the following:

  • The sample should contain urea, nonionic detergents, and IPG Buffer or carrier ampholytes.
  • Apply the sample in dilute solutions (60–100 µg protein per 100 µL).

For micropreparative applications, rehydration loading is recommended. Paper-bridge loading is recommended if using basic strips.

Attaching sample cups to the cup bar and pressing them against Immobiline® DryStrip gels

Figure 1.Attaching sample cups to the cup bar and pressing them against Immobiline® DryStrip gels

Applying sample into sample cups

Figure 2.Applying sample into sample cups.

Materials
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