Protein Gel 2D Electrophoresis Support—Troubleshooting

All lots of IEF gels that we manufacture exhibit these lines because carrier ampholytes do focus into tight bands extending across the gel and do stain a little. The intensity of these lines varies depending on the ampholytes used. One or more species may be overabundant, leading to more intense ampholyte bands. Usually these are still quite faint compared to the sample.

During IEF, it is common for the current to drop below 1 mA. Most power supplies register this as a “No Load” error and automatically shut off, resulting in the stopping of the gel run. This can be bypassed in some power supplies by disabling or turning off the “Load Check” feature.

The bubbles are probably caused by carbon dioxide outgassing from the gel. Carbon dioxide is a weak acid and will migrate into the acidic region of the IEF gel and come out of solution as it concentrates. Thorough degassing of the cathode buffer will reduce the problem. In addition, the gel can be run in the cold room so that the carbon dioxide won't come out of solution. If some bubbles form in the gel in the last few minutes of the run, they will probably not cause visible distortion. If they form earlier in the run, they may cause distortion because they act as insulators and change the gel temperature. These bubbles form between the gel surface and the cassette.

• The standards should be examined for proper focusing.
• A likely cause is a mixing of the anode and cathode buffers. Even a small amount can cause improper runs. Unlike Tris-Glycine or Tricine gels, where the inner and outer buffer chambers are filled with the same buffer, extreme caution must be taken to prevent splash-over.
• High salt concentration can cause bands to run very crookedly. Also high salt in a sample lane can affect the run of adjacent standard lanes with low salt.

It is not unusual for antibodies (even monoclonals) to be differentially glycosylated, and therefore not focus well on IEF gels. Sometimes a monoclonal can even run more smeary or unfocused than a polyclonal, over a range of pH 6.5 to 8.0. Try running the gel longer to improve the focus, but often the improvement may be minimal if any at all.

A possible reason is poor contact between electrodes or incomplete circuit. Make sure that you have added 600 μL deionized water to the Electrode Wicks and the gel is exposed at the anodic and cathodic ends of the cassette. Check the power supply. Be sure to set the ‘Load Check’ to off to enable the power supply to operate at low current.

Here are the possible causes and solutions:

• Low protein load. Increase the protein load. Use an accurate and sensitive protein estimation method.
• Improper sample preparation. Increase solubilization reagents. Use at least 8 M urea for solubilization. Add DTT and non-ionic detergents (see manual for details).
• Strip not correctly oriented. Align the strip correctly as described in the manual. Be sure to have the gel side up when loading the strip into the ZOOM IPGRunner Cassette.
• Air bubbles between the strip and 2D gel. Smooth out any air bubbles.
• Insensitive detection method. Use sensitive detection methods such as silver staining or immunoblotting.

Here are the possible causes and solutions:

• High salt concentration. Limit the salt concentration in the samples to 10 mM or less. If possible, adjust the salt concentration of your sample by ultrafiltration, dialysis, or gel filtration.
• High Power. Check power settings.
• Poor strip rehydration. Rehydrate the strips in 140 μL sample rehydration buffer for 1 hour as described in the manual. Rehydration can be extended to overnight if you use 155 μL rehydrating buffer. Make sure that the rehydration buffer is covering the strip completely.
• Liquid in the inner chamber. Do not pour any liquid or buffer in the inner chamber. Check for any leakage in the inner chamber.

Here are the possible causes and solutions:

• Improper sample preparation. Increase solubilization reagents in the rehydration buffer. Use 8 M urea for solubilization. Add DTT and non-ionic detergents as described in the manual.
• Incorrect focusing time. Increase or decrease the focusing time based on the initial results.
• High protein load. Decrease the protein load. Use an accurate and sensitive protein estimation method.
• Salt, lipids, or nucleic acid impurities present in the sample. Remove interfering substances (see manual for details).
• Poor strip rehydration. Rehydrate the strips in 140 μL rehydrating buffer for 1 hour as described in the manual. Rehydration can be extended to overnight if you use 155 μL rehydrating buffer. Make sure the rehydration buffer is covering the strip completely.

Here are the possible causes and solutions:

• Impure solutions. Use ultrapure reagents to prepare the rehydration buffer, equilibration buffer, and buffers for SDS/PAGE.
• Air bubble between the strip and 2D gel. Smooth out the air bubbles.
• Poor strip rehydration. Rehydrate the strips in 140 μL rehydrating buffer for 1 hour as described in the manual. Rehydration can be extended to overnight if you use 155 μL rehydrating buffer. Make sure that the rehydration buffer covers the strip completely.
• Protein overload. Decrease the protein concentration or lower the sample volume.
• Protein precipitates. Increase solubilization reagents in the rehydration buffer (see manual). Use appropriate strips based on the pI of the protein sample. Do not add more than 10 μL of your sample to 140 μL of rehydration buffer (see manual).

Here are the possible causes and solutions:

• Protein has got oxidized. Include DTT in the rehydration buffer and perform the alkylation step.
• Impure solutions. Use ultrapure reagents to prepare the rehydration buffer, equilibration buffer, and buffers for SDS/PAGE.

Here are the possible causes and solutions:

• Protein degradation. Add protease inhibitors during sample preparation (see manual for details).
• Different subunits. Use denaturing conditions (8 M urea).
• Incomplete equilibration. Perform equilibration as described in the manual. Increase the equilibration time.
• Protein precipitates. Increase solubilization reagents in the rehydration buffer (see manual for details. Use appropriate strips based on the pI of the protein sample.
• Low protein load. Increase the protein load. You can load up to 400 μg of fractionated protein sample per ZOOM Strip. Use an accurate and sensitive protein estimation method.
• Insensitive detection method. Use sensitive detection methods such as silver staining or immunoblotting.

Here are the possible causes and solutions:

• Staining protocol modified. Refer to the manufacturer’s recommendations for correct staining protocols. To achieve best results, be sure to follow all steps exactly as given in the protocol, especially for silver staining. Changes in the protocol can result in high background.
• Background staining due to ampholytes. Prior to staining the 2D gel, thoroughly wash the gel to remove ampholytes. Use ZOOM Carrier Ampholytes as they provide a clear background due to very low non-specific binding of dyes and stains.