Horizontal Streaking

• 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

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.

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.

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.

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.

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).

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)

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.

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.

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.

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.

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).

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)