Introduction
2-D gel electrophoresis (2DGE) is a widely used protein analysis tool. When it is applied to the separation of basic proteins (pH 7-10), achieving good resolution can be a challenge. Cup loading is an approach commonly used to improve resolution when separating basic proteins.(1) While it is not known how cup loading improves resolution, we speculate that only proteins within the pI range of the IPG strip actually enter the gel, eliminating contaminating nucleic acids, large molecules, and interfering proteins, all of which can contribute to streaking on 2-D gels and affect resolution.(2) However, cup loading alone does not address all of the resolution problems related to 2-D separation of proteins with basic pIs. Streaks and smears may still occur due to a multitude of factors.
One common cause of streaking associated with basic protein separation is disulfide bond formation in the protein sample. To eliminate disulfide bond formation at cysteine residues, reducing agents are added at several points during the normal 2DGE process. However, when studying basic proteins, the normal reducing agents are rendered ineffective. This is because reducing agents like DTT become negatively charged during isoelectric focusing and migrate away from the basic end of the IPG strip towards the anode. This allows disulfide bonds to re-form randomly intra- and intermolecularly.(3) These disulfide bonds result in minor artifacts, such as protein spots that appear poorly focused and slightly blurred. They can also cause more dramatic changes such as severe comet-like streaking from spots, the disappearance of some spots, or the appearance of new spots in different locations.(4) By preventing disulfide bond formation, these artifacts are eliminated. Prevention can be accomplished by treating the sample with a reducing agent followed by an alkylating agent. Here we discuss the resolution problems observed with basic protein separation and explain how reduction-alkylation and cup loading can be used together to mitigate streaking on 2-D gels.

Methods
Sample preparation
Protein samples were prepared from mouse liver tissue (Pel-Freez Biologicals, Rogers, AR). Tissue was minced, then extracted using a Dounce homogenizer (Wheaton Scientific, Millville, NJ) in 6 volumes of 7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris base, 2 mM TBP, and 50 mM DTT. The extract was subjected to ultrasonification using a Branson Sonifer Model 450 for 3 3 30 sec to fragment nucleic acids. After sonication, 150 U/ml of Benzonase (Sigma, St. Louis, MO) and 2 mM MgCl2 were added and the extract incubated for 30 min at room temperature to further degrade nucleic acids. The sample was clarified by centrifugation at 20,000 g for 30 min, aliquoted, frozen on dry ice, and stored at -80 C. Approximately 650 mg of protein was diluted to a volume of 150 ml with 7 M urea, 2 M thiourea, 4% CHAPS, and 0.2% ReadyStrip™ pH 7-10 buffer, then either treated according to the instructions in the ReadyPrep™ reduction-alkylation kit (Bio-Rad Laboratories, Hercules, CA) or mock treated. For both treatments, the pH of the solution was adjusted by addition of alkylation buffer, tributylphosphine (TBP) added to a final concentration of 5 mM, and reactions incubated for 30 min at room temperature. Then, either 0.5 M iodoacetamide was added to final concentration of 15 mM (+treatment) or an equal volume of water was added to mock reactions, and the samples incubated at room temperature for 60 min. The reactions were not quenched with additional TBP since the reactions contain 2 M thiourea, which is a strong quencher of iodoacetamide.(5). At the end of the reduction-alkylation or mock treatment, the protein sample was centrifuged for 5 min at 16,000 3 g to pellet any debris.

Sample application
Mouse liver protein (~600 mg) was applied to 24 cm ReadyStrip pH 7-10 IPG strips using either cup loading or passive rehydration.
Cup loading — 24 cm ReadyStrip pH 7-10 IPG strips (Bio-Rad) were rehydrated gel side down with 450 ml (10% greater volume than recommended for passive rehydration) of 7 M urea, 2 M thiourea, 4% CHAPS, 0.2% ReadyStrip pH 7-10 buffer, and 0.001% Bromophenol Blue. Each strip was covered with mineral oil and left to rehydrate overnight. The rehydrated IPG strips were removed from the rehydration tray, excess mineral oil blotted with wet filter paper, and placed gel side up into the cup loading focusing tray. Wet paper wicks were placed at both ends of the strip and a 150 ml sample cup was placed near the anode. Reduced and alkylated protein (600 mg in 150 ml) was loaded into each sample cup and overlaid with mineral oil to prevent evaporation.

Passive rehydration — Both reduced and alkylated and mock treated samples were diluted with the same buffer used for cup loading. Prior to strip rehydration, additional TBP was added to the mock-treated samples to a final concentration of 2 mM. ReadyStrip pH 7-10, 24 cm IPG strips were rehydrated gel side down overnight with 450 ml of protein sample (~600 mg) and covered with mineral oil.

2-D electrophoresis
IEF of proteins was performed using a three-step protocol and the PROTEAN® IEF cell (Bio-Rad) at a temperature of 20 C. The IPG strips were initially conditioned for 30 min at 250 V (rapid voltage ramping), linearly ramped to 10,000 V over 2 hr, and focused at 10,000 V for 90,000 V-hr. Focused strips were stored in rehydration/ equilibration trays at -80 C until second-dimension separation could be performed. For second-dimension separations, IPG strips were first equilibrated for 10 min in 8 ml of equilibration buffer (6 M urea, 2% SDS, 0.375 M Tris-HCl, pH 8.8, 20% glycerol) containing 2% DTT and then for 10 min in 8 ml of equilibration buffer containing 2.5% iodoacetamide. Strips were then transferred onto 12.5% Tris-HCl SDS-PAGE gels (23 3 25.5 cm) and electrophoresed in the PROTEAN® Plus Dodeca™ (Bio-Rad) cell for 2,800 V-hr.

Staining and imaging
After electrophoresis, gels were fixed in 30% ethanol, 10% acetic acid for 30 min and then stained for 60 min with Bio-Safe™ Coomassie stain (Bio-Rad). Gels were destained with several changes of water prior to imaging using the Bio-Rad GS-800™ calibrated densitometer and PDQuest™ 2-D analysis software.

Results
Effects of alkylating the protein sample
Using mouse liver as a test sample, reduction-alkylation treatment clearly led to improved 2-D protein separation on pH 7-10 IPG strips, significantly reducing horizontal streaking (compare B and C in figure). The number and intensity of spots increased in samples subjected to reduction-alkylation treatment. The greatest improvement was seen above pH 8, which is where intra- and inter-disulfide exchange is expected to be most prevalent.

Effects of cup loading
IEF of proteins in the basic pH range is often improved using cup loading.(6) The sample without cup loading showed elongated spots with more extensive streaking across the entire gel. The sample that was cup loaded showed more protein spots, and those spots were sharper and more robust. This was particularly so in the middle section of the gels (compare A and B in figure).
The combination of reduction-alkylation treatment and cup loading at the anode produced dramatic improvements in 2-D gel quality compared to a standard 2-D protocol using TBP reducing agent and passive rehydration (compare A and C in figure.)

Conclusions
There are two recommendations for improving large format 2-D gels, especially when targeting proteins with basic pIs. The first is alkylation of denatured proteins to prevent disulfide bond formation. The second is the use of cup loading of samples for IEF. The data indicate that both seem to independently improve spot resolution.

About the authors
Mary Grace Brubacher, M. Sci. is a Senior Product Manager at Bio-Rad Laboratories. Her efforts focus on products for proteomics, specializing in sample preparation for 2-D electrophoresis. Jessica Dines, Ph.D., Senior Scientist, and William Strong, Ph.D., Research and Development Group Leader, are also with Bio-Rad Laboratories.
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References
1. Görg, A. et al. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21:1037-1053 (2000).
2. Garfin, D. and Heerdt, L. (eds) 2-D Electrophoresis for Proteomics: A Methods and Product Manual. Bio-Rad bulletin 2651 (2000).
3. Hoving S et al., Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients. Proteomics 2:127-134 (2002).
4. Herbert, B. et al. Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when and how? Electrophoresis 22:2046-2057 (2001).
5. Galvani, M. et al. Protein alkylation in the presence/absence of thiourea in proteome analysis: a matrix assisted laser desorption/ionization-time of flight-mass spectrometry investigation. Electrophoresis 22:2066-2074 (2001).
6. Bio-Rad Laboratories. Cup loading tray for the PROTEAN IEF cell instruction manual, Bio-Rad bulletin 4006216.