Horizontal Streaking

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Overview

Welcome to the 2-D Doctor™ section on horizontal 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 horizontal streaking of 2-D gels.

Other sections in the 2-D Doctor:

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

Problem: Horizontal Streaking Across Entire Gel

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Likely Cause(s) Protein overloading
Proteins not properly and stably solubilized
DNA contamination
Incomplete focusing
Recommended Solution(s) Ensure that all proteins are completely solubilized by using a strong chaotropic extraction reagent. The concentrations of urea, thiourea, detergents, carrier ampholytes, and reducing agents (DTT or TBP) are also critical. Every sample type typically requires a new sample preparation method. A good starting point for sample preparation includes the following standard buffer solutions:
  • 8–9 M urea, 4% (w/v) CHAPS, 2 mM TBP or 1% (w/v) DTT, 40 mM Tris, 0.2% (w/v) Bio-Lyte® (ampholyte), pH 3–10
  • 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2 mM TBP or 1% (w/v) DTT, 40 mM Tris, 0.2% (w/v) Bio-Lyte (ampholyte), pH 3–10
  • ReadyPrep™ sequential extraction kit reagent 3: 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3–10, 40 mM Tris, 0.2% (w/v) Bio-Lyte (ampholyte), pH 3–10
  • ReadyPrep 2-D rehydration/sample buffer 1: 7 M urea, 2 M thiourea, 1% ASB-14, 40 mM Tris
Allow sufficient time for full denaturation and solubilization. Allow the sample to sit at room temperature in the solubilization solution for 1 hr before applying to IEF.
Remove insoluble protein complexes by centrifugation (>10,000 x g) prior to IEF.
Recommended Products ReadyPrep protein extraction kit (total protein)
ReadyPrep sequential extraction kit
 

Problem: Intermittent Horizontal Streaks

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Likely Cause Contaminants such as salts, ionic detergents (for example SDS), peptides, nucleic acids, polysaccharides, lipids, phenolic compounds
Recommended Solution(s) Different removal techniques are recommended for each class of contaminant.
Salt

Avoid salt concentrations above 40 mM to ensure high-quality IEF. Remove salts in samples by dialysis, gel filtration, protein concentration devices, or precipitation, followed by resolubilization. Precipitation can be accomplished with 10% TCA in acetone (Damerval et al. 1986) or with the ReadyPrep 2-D cleanup kit. Following precipitation, remove the precipitating agent, then resuspend the sample in an IEF/2-D sample buffer of your choice. Bio-Spin®,™/Micro Bio-Spin™ 6 columns can also remove salt with an easy-to-use spin column format.

Ionic detergents

SDS is a very effective detergent for solubilizing otherwise insoluble proteins. If SDS has been used for sample preparation, dilute the sample into a solution containing an excess of neutral or zwitterionic detergent prior to IEF. The final concentration of SDS in the sample should be 0.25% (w/v) or lower, and the ratio of the excess detergent to SDS should be at least 8:1. If this is not possible, remove SDS by protein precipitation followed by washing, then resolubilize in an IEF/2-D sample buffer of your choice, as described above for salt removal.

Nucleic acids

The presence of nucleic acids tends to increase sample viscosity and clog the pores of the polyacrylamide matrix. Enzymatic digestion with endonucleases is the most straightforward way to remove DNA. This may be done by adding a 0.1x solution containing 1 mg/ml DNase I, 0.25 mg/ml RNase A, and 50 mM MgCl2 to the sample and incubating on ice. Note that magnesium ions are required for DNase activity.

Polysaccharides

As with nucleic acids, large polysaccharides can interfere with IEF by obstructing gel pores. Remove polysaccharides by precipitating in TCA/acetone (Damerval et al. 1986), precipitating with ammonium acetate following phenol extraction (Hurkman and Tanaka 1986), or using the ReadyPrep 2-D cleanup kit.

Lipids

Proteins bind to lipids by hydrophobic interactions, giving rise to artifactual heterogeneity on 2-D gels. To break lipid-protein interactions, add excess detergent. Some lipid-rich samples may require chemical delipidation with organic solvents prior to sample resolubilization. This may be done using a mixture of chloroform and methanol (Wessel and Flugge 1984).

Phenolic compounds

Plant tissues contain a large variety of phenolic compounds that may reversibly bind polypeptides via strong hydrogen bonds. To prevent these problems:

  • Adsorb polyphenols using polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP)
  • Add reducing agents such as DTT, ascorbate, or sulfite to the sample preparation solution to prevent phenolic oxidation
  • Use thiourea, which is likely to already be present in the sample preparation solution, to inhibit the enzyme that oxidizes polyphenols
  • Prevent phenolic oxidation by disrupting liquid nitrogen-frozen tissue directly in a strongly denaturing mixture such as TCA/acetone (Damerval et al. 1986)
Recommended Products ReadyPrep 2-D cleanup kit
Bio-Spin/Micro Bio-Spin 6 columns
 

Problem: Localized Intermittent Horizontal Streaks

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Likely Cause Contaminants such as salts, ionic detergents (for example, SDS), peptides, nucleic acids, polysaccharides, lipids, phenolic compounds
Recommended Solution(s) Different removal techniques are recommended for each class of contaminant.
Salt

Avoid salt concentrations above 40 mM to ensure high-quality IEF. Remove salts in samples by dialysis, gel filtration, protein concentration devices, or precipitation, followed by resolubilization. Precipitation can be accomplished with 10% TCA in acetone (Damerval et al. 1986) or with the ReadyPrep 2-D cleanup kit. Following precipitation, remove the precipitating agent, then resuspend the sample in an IEF/2-D sample buffer of your choice.

Ionic detergents

SDS is a very effective detergent for solubilizing otherwise insoluble proteins. If SDS has been used for sample preparation, dilute the sample into a solution containing an excess of a neutral or zwitterionic detergent prior to IEF. The final concentration of SDS in the sample should be 0.25% (w/v) or lower, and the ratio of the excess detergent to SDS should be at least 8:1. If this is not possible, remove SDS by protein precipitation followed by washing, then resolubilize in an IEF/2-D sample buffer of your choice, as described above for salt removal.

Nucleic acids

The presence of nucleic acids tends to increase sample viscosity and clog the pores of the polyacrylamide matrix. Enzymatic digestion with endonucleases is the most straightforward way to remove DNA. This may be done by adding a 0.1x solution containing 1 mg/ml DNase I, 0.25 mg/ml RNase A, and 50 mM MgCl2 to the sample and incubate on ice. Note that magnesium ions are required for DNase activity.

Polysaccharides

As with to nucleic acids, large polysaccharides can interfere with IEF by obstructing gel pores. Remove polysaccharides by precipitating in TCA/acetone (Damerval et al. 1986), precipitating with ammonium acetate following phenol extraction (Hurkman and Tanaka 1986), or using the ReadyPrep 2-D cleanup kit.

Lipids

Proteins bind to lipids by hydrophobic interactions, giving rise to artifactual heterogeneity on 2-D gels. To break lipid-protein interactions, add excess detergent. Some lipid-rich samples may require chemical delipidation with organic solvents prior to sample resolubilization. This may be done using a mixture of chloroform and methanol (Wessel and Flugge 1984).

Phenolic compounds

Plant tissues contain a large variety of phenolic compounds that may reversibly bind polypeptides via strong hydrogen bonds. To prevent these problems:

  • Adsorb polyphenols using polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP)
  • Add reducing agents such as DTT, ascorbate, or sulfite to the sample preparation solution to prevent phenolic oxidation
  • Use thiourea, which is likely to already be present in the sample preparation solution, to inhibit the enzyme that oxidizes polyphenols
  • Prevent phenolic oxidation by disrupting liquid nitrogen-frozen tissue directly in a strongly denaturing mixture such as TCA/acetone (Damerval et al. 1986)
Recommended Products ReadyPrep 2-D cleanup kit
Bio-Spin/Micro Bio-Spin 6 columns
 

Problem: Concentrated Regional Horizontal Streaks

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Likely Cause Protein overloading
Recommended Solution(s) Quantitate the sample protein prior to IEF to ensure that the protein has the proper concentration. 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.
Dilute the sample.
Use a longer IPG strip and larger gel size to allow for a greater protein load.
If possible, selectively remove high-abundance proteins. For example, in serum, albumin makes up 70–90% of total serum protein. Loading the recommended amount of protein onto the strip can obscure the many other proteins present. Removing the albumin will effectively increase the total protein load of other proteins, minimizing streaking and enhancing resolution of low-abundance proteins.
Recommended Products RC DC protein assay
Aurum serum protein and Aurum Affi-Gel® Blue mini kits
ProteoMiner protein enrichment kits
Rotofor®, Mini Rotofor, and MicroRotofor™ cells
Model 491 prep cell and mini prep cell
 

Problem: Regional Horizontal Streaks

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Likely Cause Protein overloading
Recommended Solution(s) Quantitate the sample protein prior to IEF to ensure that the protein has the proper concentration. 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.
Dilute the sample.
Use a longer IPG strip and larger gel size to allow for a greater protein load.
If possible, selectively remove high-abundance proteins. For example, in serum, albumin makes up 70–90% of total serum protein. Loading the recommended amount of protein onto the strip can obscure the many other proteins present. Removing the albumin will effectively increase the total protein load of other proteins, minimizing streaking and enhancing resolution of low-abundance proteins.
Recommended Products RC DC protein assay
Aurum serum protein and Aurum Affi-Gel Blue mini kits
ProteoMiner protein enrichment kits
Rotofor, Mini Rotofor, and MicroRotofor cells
Model 491 prep cell and mini prep cell
 

Problem: Horizontal Spot Streaking

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Likely Cause IEF not optimized
Recommended Solution(s) Optimize the sample focusing time by running a time course. With the PROTEAN® i12™ IEF system, run the same sample on multiple strips and program different run durations. For a traditional IEF system, run the sample on multiple IPG strips and remove strips after different time points (20 kV-hr, 30 kV-hr, 40 kV-hr, etc.).
If using a traditional IEF system, check that samples within the run are similar. Conventional IEF cells set a total current limit for the whole tray, so if one particular sample is more conductive, it will draw most of the current and decrease the focusing rate of the other strips in the tray. Therefore, samples with very different conductivities should be run separately. The PROTEAN i12 IEF system can be used to optimize sample conditions and run multiple different samples at once.
Recommended Products PROTEAN i12 IEF system
ReadyStrip IPG strips
 

Problem: Extensive Horizontal Spot Streaking

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Likely Cause IEF not optimized
Recommended Solution(s) Optimize the sample focusing time by running a time course. With the PROTEAN i12 IEF system, run the same sample on multiple strips and program different run durations. For a traditional IEF system, run the sample on multiple IPG strips and remove strips after different time points (20 kV-hr, 30 kV-hr, 40 kV-hr, etc.).
If using a traditional IEF system, check that samples within the run are similar. Conventional IEF cells set a total current limit for the whole tray, so if one particular sample is more conductive, it will draw most of the current and decrease the focusing rate of the other strips in the tray. Therefore, samples with very different conductivities should be run separately. The PROTEAN i12 IEF system can be used to optimize sample conditions and run multiple different samples at once.
Recommended Products PROTEAN i12 IEF cell
ReadyStrip IPG strips
 

Problem: Horizontal Spot Streaking in Basic Region

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Likely Cause DTT depletion, reoxidation of disulfide bonds
Recommended Solution(s) Historically, DTT was used in the sample rehydration and equilibration solutions to cleave disulfide bonds and maintain them in a reduced state. However, DTT is deprotonated and negatively charged at an alkaline pH and will migrate out of the basic range in an IPG strip, allowing reoxidation of thiol groups and the formation of inter and intramolecular disulfide bonds.

Reduce and alkylate the sample prior to IEF with tributylphosphine (TBP) and iodoacetamide (Herbert et al. 2001). This will help prevent disulfide bonds from re-forming, which is especially problematic for basic proteins due to the increased rate of disulfide bond formation in alkaline environments. The ReadyPrep reduction-alkylation kit eliminates the potential for disulfide bond formation.

 

Enhance separation of basic proteins by cup loading the sample at the anode after rehydration of the IPG strip with IEF buffer.
Recommended Products ReadyPrep reduction-alkylation kit
i12 Sample cup holder
 

Problem: Localized Horizontal Spot Streaking

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Likely Cause(s) Insufficient rehydration solution
Improper IPG strip rehydration
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.
 

 

References

Damerval C et al. (1986). Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electrophoresis 7, 52–54.

Herbert B et al. (2001). Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when, and how? Electrophoresis 22, 2046–2057.

Hurkman WJ and Tanaka CK (1986). Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol 81, 802–806.

Wessel D and Flugge UI (1984). A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138, 141–143.

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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
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
3098 Expression Proteomics Overview 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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