Irregular Spot Patterns

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Overview

Welcome to the 2-D Doctor™ section on irregular spot patterns. The 2-D Doctor is a self-help guide that enables you to troubleshoot your 2-D gel issues. Here you will find solutions to the problem of irregular spot patterns on 2-D gels.

Other sections in the 2-D Doctor:

Problems and Solutions

Click on the thumbnail that is most representative of your own gel to find out the probable cause of and specific solutions to your problem.

 

For additional help, you can also view videos and browse FAQs.

Problem: Wavy Spots

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Likely Cause Insufficient overlay solution after gel casting
Recommended Solution Overlay the gel with water-saturated butanol (n-butanol, l-butanol, or t-butanol) or t-amyl alcohol immediately after gel casting. These ensure that the gel has a clean, straight top edge. Use the overlay recommended by the manufacturer of the electrophoresis cell.
Recommended Product Bio-Rad precast gels
 

Problem: Local Wavy Disturbance of Spots

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Likely Cause(s) SDS gel not evenly polymerized
SDS gel cassette leaking
SDS gel cassette leaking during casting
Recommended Solution(s) Optimize the APS and TEMED concentrations.
De-gas solutions prior to the addition of APS/TEMED.
Perform casting at room temperature, warming the glass plates if necessary. Be aware that the polymerization process is temperature dependent. If the temperature is too low, polymerization may be compromised.
Recommended Product Bio-Rad precast gels
 

Problem: Missing High Molecular Weight Proteins

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Likely Cause(s) Proteolysis
Low-quality or old reagents
Large proteins not entering IPG strips during rehydration
Large proteins not entering the second-dimension gels during electrophoresis
Recommended Solution(s) Include protease inhibitors in sample preparation buffers.
Use a combination of thiourea and urea in the sample preparation buffer.
Use fresh reagents.
Use a different rehydration method or rehydration buffer for better solubility.
Increase equilibration time.
Recommended Products ReadyPrep™ total protein extraction kit
MicroRotofor™ cell lysis kits
ReadyPrep 2-D starter kit equilibration buffer I
ReadyPrep 2-D starter kit equilibration buffer II
Bio-Rad iodoacetamide
 

Problem: Vertically Doubled Spots

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Likely Cause(s) IPG strip improperly placed on SDS gel
Temperature gradient occurring in the gel during the second-dimension gel run
Low-quality or old reagents
Improper equilibration
Polypeptides not sufficiently reduced
Recommended Solution(s) Ensure that the IPG strip is in direct contact with the second-dimension gel along its entire length. To prevent twisting of the IPG strip during placement, press the plastic backing of the IPG strip firmly against one of the 2-D gel plates. Use staggered plates, as they allow easier placement of the IPG strip.
Reduce the maximum current. Refer to the instruction manual for the electrophoresis cell for recommended running conditions.
Equilibrate the IPG strips with the recommended volume of buffer for the recommended amount of time, or longer if necessary.
Use fresh reagents, including Iodoacetamide and DTT.
Use a better circulation system for even heat dissipation in the electrophoresis cell during a run.
Recommended Products Bio-Rad precast gels
Bio-Rad glass plates
Bio-Rad gel casting chambers
Bio-Rad iodoacetamide
Bio-Rad dithiothreitol, 1g
Bio-Rad dithiothreitol, 5g
 

Problem: Diffuse and Uneven Background in Silver-Stained Gel

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Likely Cause(s) Insufficient washing steps
Insufficient fixative steps
Contaminant in agarose overlay solution or running buffer
Recommended Solution(s) Ensure that the gels are fully immersed and floating properly in the staining solution. They should not stick to the staining tray. Do not place too many gels in one tray.
Perform more washing steps if needed. Ensure that the water used is of high quality, and that the staining tray is clean.
Some uneven background stain is normal when using a silver stain. Due to the different chemicals and ions migrating into the gels, some regions can be stained with different colors or intensities. Often, a longer fixing procedure can minimize this effect.
If you are still experiencing difficulty, use a different stain, such as Flamingo™ fluorescent gel stain or Oriole™ fluorescent gel stain.
Recommended Products Dodeca stainer
Dodeca silver stain kit
Silver Stain Plus™ kit
Flamingo fluorescent gel stain
Oriole fluorescent gel stain
 

Problem: Localized Diffuse & Uneven Background in Silver-Stained Gel

Likely Cause(s) Insufficient washing steps
Insufficient fixative steps
Contaminant in agarose overlay solution or running buffer
Recommended Solution(s) Ensure that the gels are fully immersed and floating properly in the staining solution. They should not stick to the staining tray. Do not place too many gels in one tray.
Perform more washing steps if needed. Ensure that the water used is of high quality, and that the staining tray is clean.
Some uneven background stain is normal when using a silver stain. Due to the different chemicals and ions migrating into the gels, some regions can be stained with different colors or intensities. Often, a longer fixing procedure can minimize this effect.
If you are still experiencing difficulty, use a different stain, such as Flamingo fluorescent gel stain or Oriole fluorescent gel stain.
Recommended Products Dodeca stainer
Dodeca silver stain kit
Silver Stain Plus kit
Flamingo fluorescent gel stain
Oriole fluorescent gel stain
 

Related Content

2-D Electrophoresis: Avoiding Horizontal Streaks
Horizontal streaks in 2-D gels are the result of poor protein separation during isoelectric focusing (IEF). This tutorial discusses the most common sources of horizontal streaks in 2-D gels: problems with sample preparation and inappropriate IEF conditions.
 
2-D Video Tutorial
How to run a 2-D Gel from start to finish.
 
Literature
Number Description Download
2651 2-D Electrophoresis for Proteomics: A Methods and Product Manual, Rev F Click to download
2587 High-Performance 2-D Gel Electrophoresis Using Narrow pH-Range ReadyStrip IPG Strips, Rev C Click to download
2670 Separation and Comparison of Proteins From Virulent and Nonvirulent Strains of the Fish Pathogen Flavobacterium psychrophilum, Using a 2-D Electrophoretic Approach Click to download
2740 Use of the PROTEAN Plus Dodeca Cell for Second-Dimension SDS-PAGE, Rev A Click to download
2778 Focusing Strategy and Influence of Conductivity on Isoelectric Focusing in Immobilized pH Gradients, Rev A Click to download
2859 Combination of 2-D Gel and Liquid-Phase Electrophoretic Separations As Proteomic Tools in Neuroscience, Rev A Click to download
3098 Expression Proteomics Overview Brochure, Rev B Click to download
5545 Sensitivity and Protein-to-Protein Consistency of Flamingo Fluorescent Gel Stain Compared to Other Fluorescent Stains (Poster), Rev A Click to download
6097 PROTEAN i12 IEF System Brochure, Rev A Click to download
2621 PROTEAN Plus Dodeca Cell System Brochure, Rev C Click to download
3095 2-D Electrophoresis: Tools for Rapid, High-Resolution Protein Separations Brochure, Rev B Click to download
3096 Sample Preparation: Tools for Protein Sample Extraction, Cleanup, Fractionation, and Depletion Brochure, Rev B Click to download
3097 Imaging and Analysis: Tools for Acquisition and Analysis of Protein Expression Data Brochure, Rev B Click to download
Number Description Options
6222 IPG Equilibration for the Second Dimension, Placement and Agarose Embedding of IPG Strips
Click to download
6236 Using Precison Plus Protein Standard Plugs
Click to download
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