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This troubleshooting table lists problems that may be encountered in two-dimensional difference gel electrophoresis (2D DIGE) results. The table includes descriptions of possible causes of poor results and offers methods to prevent problems in future experiments.

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Symptom	Possible cause	Remedy
No distinct spots are visible	Sample is insufficient. Insufficient sample entered the Immobiline^® DryStrip gel due to poor sample solubilization.	Increase the amount of sample applied. Increase the concentration of the solubilizing components in the sample solution (see section 1.6).
Sample contains impurities that prevent focusing.	Increase the focusing time or modify the sample preparation method.
The pH gradient is incorrectly oriented.	The “+” end of the Immobiline^® DryStrip is the acidic end and should point toward the anode (+).
(Flatbed gel format) Immobiline^® DryStrip gel is placed wrong side down on second-dimension gel.	Ensure that the Immobiline^® DryStrip gel 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. Prepare fresh staining solutions.	Check expiry dates on 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 °C, as cyanate, a urea degradation product, will carbamylate proteins, changing their pI.
Protein oxidation.	Use DeStreak Rehydration Solution. During equilibration, add DTT in first step to reduce the disulfide. Add iodoacetamide in the second step to alkylate the thiol groups to prevent proteins from reoxidizing.
Distortion of 2-D pattern $\"Distortion$	(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 1-butanol.
(Vertical gel format) Uneven polymerization of gel due to incomplete polymerization, too rapid polymerization, or leakage during gel casting.	Degas the gel solution. \r\n \r\nPolymerization can be accelerated by increasing by 50% the amount of ammonium persulfate and TEMED used. Polymerization can be slowed by decreasing by 33% the amount of ammonium persulfate and TEMED used. \r\n \r\nEnsure that there is no leakage during gel casting.
Horizontal streaking or incompletely focused spots (anodic sample application, in which the problem is visible at the anodic end of the IPG strip) $\"Horizontal$	Sample applied at too acidic pH.	Increase the concentration of IPG buffer in sample and Immobiline^® DryStrip. Add slightly more alkaline IPG buffer to the sample. \r\n \r\nApply the sample at the cathode. \r\n \r\nNote: Repeated precipitation resolubilization cycles produce or increase horizontal streaking. \r\n \r\nSee section 1.6 (Composition of sample preparation solution) for general guidelines for sample solubilization.
Horizontal streaking or incompletely focused spots (rehydration loading) $\"Horizontal$	Sample is poorly soluble in rehydration solution.	Increase the concentration of the solubilizing components in the rehydration solution (see section 2.6, Components of rehydration solution). \r\n \r\nIncrease concentration of IPG Buffer.
Underfocusing. Focusing time was not long enough to achieve steady-state focusing.	Prolong focusing time.
Horizontal streaking or incompletely focused spots (all sample application methods) $\"Horizontal$	Interfering substances. Non-protein impurities in the sample can interfere with IEF, causing horizontal streaking.	Modify sample preparation to limit these contaminants. \r\n \r\nUse 2-D Clean-Up Kit (section 1.4.1, Cleaning up samples using 2-D Clean-Up Kit). \r\n \r\nThe effect of ionic impurities can be reduced by modifying the IEF protocol. Limit the voltage to 100–150 V for 2 h, then resume a normal voltage step program. This allows the ions in the sample to move to the ends of the Immobiline^® DryStrip gel.
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 nonionic detergent present must be at least eight times higher than the concentration of any ionic detergent to ensure complete removal of SDS from the protein.
Horizontal stripes across gel $\"Horizontal$	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 Immobiline^® DryStrip gels and sample cups) $\"Prominent$	(Flatbed gel format) Sample aggregation or precipitation.	Dilute the sample and apply as a larger volume. \r\n \r\nProgram a low initial voltage and increase voltage gradually.
Vertical gap in 2-D pattern $\"Vertical$	Impurities in sample.	Modify sample preparation.
Impurities in rehydration solution components.	Use only high-quality reagents. \r\n \r\nDeionize urea solutions.
Bubble between Immobiline^® DryStrip gel and top surface of second-dimension gel.	Ensure that no bubbles are trapped between the Immobiline^® DryStrip gel and the top surface of the second-dimension gel.
(Flatbed gel format) Urea crystals on the surface of the Immobiline^® DryStrip gel.	Allow residual equilibration solution to drain from the Immobiline^® DryStrip gel before placing the strip on the second-dimension gel.
(Flatbed gel format) Bubbles under the Immobiline^® DryStrip gel.	Ensure that the Immobiline^® DryStrip gel is placed firmly on the gel with no air bubbles trapped underneath. Stroke the plastic backing of the Immobiline^® DryStrip gel gently with a pair of forceps to remove trapped bubbles.
Poor representation of higher-molecular-weight proteins	Proteolysis of sample.	Prepare sample in a manner that limits proteolysis and/or use protease inhibitors (Protease inhibition using Protease Inhibitor Mix).
Insufficient equilibration.	Prolong equilibration time.
Poor transfer of protein from Immobiline^® DryStrip gel 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 (Table 18, Using DeStreak Rehydration Solution).
Point streaking $\"Point$	(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 Immobiline^® DryStrip gel 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. Prolong fixing time.
Background smear toward \r\n top of gel	(Silver staining) Nucleic acids in sample.	Add DNase and RNase to hydrolyze nucleic acids. \r\n \r\nNote: The proteins DNase and RNase may appear on the 2-D map.
High background in top \r\n region of gel	Protein contaminant in SDS electrophoresis buffer or dirty electrophoresis unit.	Make fresh SDS electrophoresis buffer. \r\n \r\nClean electrophoresis unit.

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