| Symptom | Possible cause | Remedy |
| No distinct spots are visible | Sample is insufficient. | Increase the amount of sample applied. |
 | Insufficient sample entered the IPG strip due to poor sample solubilization. | Increase the concentration of the solubilizing components in the sample solution. (See section 2.5, "Composition of sample solution.") |
| Sample contains impurities that prevent focusing. | Increase the focusing time or modify the sample preparation method. (See "Part I. Sample Preparation.") |
| The pH gradient is wrongly oriented. | The pointed end of the Immobiline™ DryStrip is the acidic end and should point toward the anode (+). |
 | (Flatbed gel format) IPG strip is placed wrong side down on second-dimension gel. | Ensure that the IPG strip is placed gel-side down (plastic backing upward) on the SDS second-dimension gel. |
| Detection method was not sensitive enough. | Use another detection method (e.g., silver staining instead of Coomassie blue staining). |
| Failure of detection reagents. | Check expiration dates on staining solutions.
Prepare fresh staining solutions. |
| Individual proteins appear as multiple spots or are missing, unclear, or in the wrong position | Protein carbamylation. | Do not heat any solutions containing urea above 30 ºC, as isocyanate, a urea degradation product, will carbamylate proteins, changing their pI. |
| Protein oxidation. | DTT in the rehydration and equilibration solutions keeps the disulphide bonds reduced. For additional protection include an iodoacetamide treatment during equilibration prior to the second-dimension separation.
Iodoacetamide alkylates the thiol groups to prevent the reduced proteins from reoxidizing. |
Spots are vertically doubled, or "twinned"
 |
(Vertical gel format) IPG strip is not placed properly. |
Ensure that the plastic backing of the IPG strip is against the glass plate on the second-dimension gel. |
Distortion of 2-D pattern
 |
(Vertical gel format) The top surface of the second-dimension gel is not flat. |
Immediately after pouring the gel, overlay the surface with water-saturated butanol. |
 | (Vertical gel format) Uneven polymerization of gel due to incomplete polymerization, too rapid polymerization, or leakage during gel casting. | Degas the gel solution.
Polymerization can be accelerated by increasing by 50% the amount of ammonium persulphate and TEMED used. Polymerization can be slowed by decreasing by 33% the amount of ammonium persulphate and TEMED used.
Ensure that there is no leakage during gel casting. |
 | (Flatbed gel format) Moisture on the surface of the second-dimension gel. | Allow ExcelGel to dry for about 5 minutes after removing plastic cover and before applying buffer strips and IPG strip. |
 | (Flatbed gel format) IPG strip not removed during electrophoresis. | Remove the IPG strip and application pieces from the second-dimension gel when the bromophenol blue dye from has moved away from the IPG strip by 4-6 mm. |
 | (Flatbed gel format) Air bubbles under the second-dimension gel cause uneven migration due to poor heat transfer. | Ensure that no bubbles are trapped under the second-dimension gel during placement on the cooling plate. |
| (Flatbed gel format) Water drops or pieces of buffer strip on the surface of the second dimension gel. | Take care that nothing is dropped or splashed onto the surface of the second-dimension gel. |
Horizontal streaking or incompletely focused spots
 |
Sample not completely solubilized prior to application. |
Be sure that the sample is completely and stably solubilized.
Note: Repeated precipitation-resolubilization cycles produce or increase horizontal streaking.
See section 2.5, "Composition of the sample solution," for general guidelines for sample solubilization. |
| Sample is poorly soluble in rehydration solution. | Increase the concentration of the solubilizing components in the rehydration solution. (See section 3.4, "IPG strip rehydration solution.")
Increase concentration of IPG Buffer. |
 | Interfering substances. Non-protein impurities in the sample can interfere with IEF, causing horizontal streaking in the final 2-D result, particularly toward the acidic side of the gel. | Modify sample preparation to limit these contaminants. (See section 2.4, "Removal of contaminants that affect 2-D results.") |
 | Ionic impurities in sample. | Reduce salt concentration to below 10 mm by dilution or desalt the sample by dialysis. Precipitation with TCA and acetone and subsequent resuspension is another effective desalting technique that removes lipids, nucleotides and other small molecules. Note: Specific and non-specific losses of proteins can occur with dialysis, gel chromatography, and precipitation/resuspension of samples.
If the sample preparation cannot be modified, the effect of ionic impurities can be reduced by modifying the IEF protocol. Limit the voltage to 100-150 V for 2 hours, then resume a normal voltage step program. This pre-step allows the ions in the sample to move to the ends of the IPG strip. |
| Ionic detergent in sample. | If the ionic detergent SDS is used in sample preparation, the final concentration must not exceed 0.25% after dilution into the rehydration solution. Additionally, the concentration of the non-ionic detergent present must be at least 8 times higher than the concentration of any ionic detergent to ensure complete removal of SDS from the proteins. |
 | High sample load. | Extend focusing time.
Load less sample.
Micropreparative separations require clean sample. Modify sample preparation to limit contaminants. (See section 2.4, "Removal of contaminants that affect 2-D results.")
Program a low initial voltage and increase voltage gradually. |
 | Underfocusing. Focusing time was not long enough to achieve steady state focusing. | Prolong focusing time. |
 | Overfocusing. Extended focusing times (over 100,000 Vh) may result in electroendosmotic water and protein movement, which can produce horizontal smearing. | Reduce focusing time. |
Horizontal stripes across gel
 | Impurities in agarose overlay or equilibration solution. | Prepare fresh agarose overlay and equilibration solution. |
Prominent vertical streak at the point of sample application (when loading IPG strips using sample cups)
 | (Flatbed gel format) Sample aggregation or precipitation. | Dilute the sample and apply as a larger volume.
Program a low initial voltage and increase voltage gradually. |
| Vertical streaking | Insufficient equilibration. | Prolong equilibration time. |
| (Flatbed gel format) Electroendosmosis. | Add 30% glycerol and 6 M urea to the SDS equilibration buffer.
Place application pieces at the end of the strips during second-dimension electrophoresis to absorb excess water. |
| Second-dimension buffer solutions prepared incorrectly. | Prepare fresh solutions. |
| Insufficient SDS in SDS electrophoresis buffer. | Use 0.1% w/v SDS. |
Vertical gap in 2-D pattern
 | Impurities in sample. | Modify sample preparation. (See section 2.4, "Removal of contaminants that affect 2-D results.") |
 | Impurities in rehydration solution components. | Use only high-quality reagents.
De-ionize urea solutions |
 | Bubble between IPG strip and top surface of second-dimension gel. | Ensure that no bubbles are trapped between the IPG strip and the top surface of the second-dimension gel. |
| (Flatbed gel format) Urea crystals on the surface of the IPG strip. | Allow residual equilibration solution to drain from the IPG strip before placing the strip on the second-dimension gel. |
| (Flatbed gel format) Bubbles under the IPG strip. | Ensure that the IPG strip is placed firmly on the gel with no air bubbles trapped underneath. Stroke the plastic backing of the IPG strip gently with a pair of forceps to remove trapped bubbles. |
Vertical regions of poor focusing
 | The IPG strip was not fully rehydrated. | Ensure that the IPG strips are rehydrated with a sufficient volume of rehydration solution.
Remove any large bubbles trapped under the IPG strip after rehydration solution is applied.
Check that the rehydration solution is evenly spread along the entire length of the IPG strip. |
| Poor representation of higher molecular weight proteins | Proteolysis of sample. | Prepare sample in a manner that limits proteolysis and/or use protease inhibitors. (See section 2.2, "Protection against proteolysis.") |
| Insufficient equilibration. | Prolong equilibration time. |
| Poor transfer of protein from IPG strip to second-dimension gel. | Employ a low current sample entry phase in the second-dimension electrophoresis run. |
| Poor entry of sample protein during rehydration. | Use recommended volume of rehydration solution. (See Tables 10 and 15.) |
Point streaking
 | (Silver staining). Dirty plates used to cast gel or particulate material on the surface of the gel. DTT and other thiol reducing agents exacerbate this effect. | Properly wash glass plates. Scavenge any excess or residual thiol reducing agent with iodoacetamide before loading the IPG strips onto the second-dimension gel. |
| Background smear toward bottom of gel | (Silver or Coomassie blue staining) Staining of carrier ampholytes. | Use IPG Buffer as carrier ampholyte mixture. Reduce concentration if necessary. |
| Background smear toward top of gel | (Silver staining) Nucleic acids in sample. | Add DNase and RNase to hydrolyze nucleic acids. Note: The proteins DNase and RNase may appear on the 2-D map. |
High background in top region of gel
 | Protein contaminant in SDS electrophoresis buffer or dirty electrophoresis unit. | Make fresh SDS electrophoresis buffer.
Clean electrophoresis unit. |
