Welcome to the 2-D Doctor™ section on vertical streaking. 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 vertical streaking of 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: Vertical Streaking at the End of the Gel
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| Likely Cause | Protein aggregation or precipitation | ||||||||||||||||||||
| Recommended Solution(s) | Dilute the sample. | ||||||||||||||||||||
| Perform a protein assay prior to IEF to ensure correct protein load. The total amount of protein that should be loaded onto an IPG strip usually depends on the length of the strip and the stain that will be used to visualize the results. | |||||||||||||||||||||
| A distinct vertical protein front can often be observed in the middle range of 2-D gels when using cup loading. During focusing, all proteins move out of the sample cup in the same direction, causing a distinct vertical band. Increase the total volt-hours to minimize this effect and attain more complete focusing. | |||||||||||||||||||||
| Load your sample using in-gel rehydration. | |||||||||||||||||||||
Prolong the time on the initial low-voltage steps and increase the voltage gradually. For example:
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Problem: Vertical Spot Streaking
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| Likely Cause(s) | Iodoacetamide is too old |
| Equilibration time was too short | |
| SDS gel has wrong pH | |
| SDS concentration was too low in equilibration solution | |
| Recommended Solution(s) | Make sure all reagents are fresh, including Iodoacetamide and DTT. |
| Prolong the equilibration step. Carry out equilibration for as long as 40 min if necessary. | |
| Ensure that the pH of the Tris buffer used for SDS-gel casting is 8.8. The wrong pH decreases the mobility of the protein-SDS complexes and causes vertical streaks of protein. | |
| Ensure that the SDS concentration is at least 2% (w/v) in the equilibration solution. | |
| Recommended Products | Bio-Rad precast gels |
| ReadyPrep™ 2-D starter kit equilibration buffer I | |
| ReadyPrep 2-D starter kit equilibration buffer II | |
| Resolving gel buffer (1.5 M Tris-HCl, pH 8.8) | |
| Bio-Rad iodoacetamide, 30g | |
| Bio-Rad dithiothreitol, 1g | |
| Bio-Rad dithiothreitol, 5g |
Problem: Vertical Streaking Down Length of Gel
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| Likely Cause(s) | Improper IPG strip rehydration |
| Insufficient rehydration solution | |
| Recommended Solution(s) | Ensure rehydration volumes are correct for the IPG strip length being used. If loading protein onto the IPG strip by including the sample in the rehydration solution, do not exceed the recommended volume, as doing so may result in incomplete entry of proteins into the IPG strip. |
| If the sample appears unevenly distributed, or if areas of the strip are not wetted with sample, slide the strip back and forth several times along the length of the channel in the focusing tray. |
Problem: Isolated Vertical Blank Stripe
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| Likely Causes | Air bubbles trapped between the IPG strip and second-dimension gel |
| Recommended Solution(s) | Ensure that the 2-D gel has a straight, level top edge and that the IPG strip is in direct contact with the 2-D gel along its entire length. |
| Use a 0.5% agarose overlay solution to prevent the IPG strip from coming loose or moving. To minimize the number of bubbles in the overlay, melt the agarose overlay solution completely prior to loading. | |
| Recommended Products | PROTEAN® Plus overlay agarose |
| ReadyPrep overlay agarose |
Problem: Vertical Streaking Across Entire Gel
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| Likely Cause(s) | Leaking of the upper buffer reservoir (cathode) of a vertical electrophoresis unit |
| Improper equilibration | |
| Old DTT and Iodoacetamide preparations | |
| Recommended Solution(s) | Prior to inserting the gel(s) into the vertical electrophoresis cell, wet the gaskets of the electrophoresis chamber with water or use a small amount of vacuum grease. |
| Increase the equilibration time to 15 min. | |
| Use fresh reagents for the equilibration step. | |
| Recommended Products | Resolving gel buffer (1.5 M Tris-HCl, pH 8.8) |
| ReadyPrep 2-D starter kit equilibration buffer I | |
| ReadyPrep 2-D starter kit equilibration buffer II | |
| Bio-Rad iodoacetamide, 30g | |
| Bio-Rad dithiothreitol, 1g | |
| Bio-Rad dithiothreitol, 5g |
Problem: Large Vertical Blank Stripe
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| Likely Cause(s) | Interfering substances; impurities in the rehydration/sample solution |
| Air bubble trapped between the IPG strip and second-dimension gel | |
| Recommended Solution(s) | Remove contaminants such as salts. |
| Use high-quality reagents and chemicals for electrophoresis to minimize the risk of impurities. Replace chemicals of questionable or unknown shelf life, origin, or quality, as these products can also contribute to poor 2-D results. | |
| Ensure no bubbles are trapped between the IPG strip and the gel surface. | |
| Recommended Products | ReadyPrep 2-D cleanup kit |
| ReadyPrep 2-D starter kit rehydration/sample buffer | |
| ReadyPrep protein extraction kit (total protein) | |
| ReadyPrep reduction-alkylation kit |
Page Contents
Videos
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.
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.
Documents
Number Description Options
2651
2-D Electrophoresis Workflow How-To Guide, Rev F
2587
High-Performance 2-D Gel Electrophoresis Using Narrow pH-Range ReadyStrip IPG Strips, Rev C
2670
Separation and Comparison of Proteins From Virulent and Nonvirulent Strains of the Fish Pathogen Flavobacterium psychrophilum, Using a 2-D Electrophoretic Approach
2740
Use of the PROTEAN Plus Dodeca Cell for Second-Dimension SDS-PAGE, Rev A
2778
Focusing Strategy and Influence of Conductivity on Isoelectric Focusing in Immobilized pH Gradients, Rev A
2859
Combination of 2-D Gel and Liquid-Phase Electrophoretic Separations As Proteomic Tools in Neuroscience, Rev A
3098
Expression Proteomics Overview Brochure, Rev B
5545
Sensitivity and Protein-to-Protein Consistency of Flamingo Fluorescent Gel Stain Compared to Other Fluorescent Stains (Poster), Rev A
6097
PROTEAN i12 IEF System Brochure, Ver B
2621
PROTEAN Plus Dodeca Cell System Brochure, Rev C
3095
2-D Electrophoresis: Tools for Rapid, High-Resolution Protein Separations Brochure, Rev B
3096
Sample Preparation: Tools for Protein Sample Extraction, Cleanup, Fractionation, and Depletion Brochure, Rev B
3097
Imaging and Analysis: Tools for Acquisition and Analysis of Protein Expression Data Brochure, Rev B
TEST
Number Description Options
| 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 |