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2D Gel Electrophoresis
Two-dimensional gel electrophoresis (2DE) is an established technique for high-resolution profiling of complex protein mixtures. 2DE can resolve thousands of protein “spots” on a single gel and is considered a key method in proteomics research. It is widely applied in protein expression profiling experiments to identify changes in protein expression levels resulting from a disease state or drug treatment. 2DE is also a valuable tool to study post-translational modifications and identify protein isoforms. Small changes in the protein mass or isoelectric point (pI) translate into a detectable protein shift.
2D electrophoresis consists of two separation steps. In the first-dimension, proteins are separated according to their isoelectric point (pI)—the pH at which the protein net charge is zero—in a process known as isoelectric focusing (IEF). During conventional IEF, amphoteric molecules, like proteins, migrate until they reach a region where the pH of the matrix matches their isoelectric point and they “focus” into sharp bands.
Novex IEF Gels can be used to determine the pI or to detect minor changes in a protein due to deamination, phosphorylation, or glycosylation. They are also excellent for native, nondenaturing applications using soluble proteins.
Immobilized pH gradient (IPG) represents an evolution of the technique. An immobilized pH gradient is produced in the gel before the sample is applied, resulting in improved resolution and reproducibility. The second dimension consists of a conventional SDS-PAGE electrophoretic separation where proteins are differentiated based on their molecular weights. 2-DE is a robust method in which the resolution acquired during the first dimension separation is preserved during the second dimension.
ZOOM IPGRunner System
Analyzing complex protein samples using traditional two-dimensional electrophoresis methods is tedious and time-consuming. The ZOOM IPGRunner System offers an integrated solution in a mini gel format. This innovative system makes sensitive protein profiling accessible to both novice and experienced proteomics researchers. The oil-free ZOOM IPGRunner System supports rapid experiment setup and execution and is a low-cost and time-saving solution for establishing the initial experimental conditions prior to running large-format 2D gel analysis.
You can obtain high-resolution results quickly and easily. The ZOOM IPGRunner System offers:
- First dimension separation in as little as 3 hours
- A unique design that eliminates messy mineral oil
- Easy-to-handle IPG strips in wide and narrow one-unit pH ranges
- Self-contained cassettes that simplify rehydration and focusing
- High performance NuPage Bis-Tris gels provide a neutral pH environment for the second dimension that reduce in-gel protein degradation.
- All components and reagents needed to get started with 2D electrophoresis are included in our first and second dimension welcome packs including PowerEase Touch HV Power Supply. Just add the IPG strips and ampholytes of your choice
Sample preparation
Sample preparation is a fundamental step in any protein analysis workflow, but it is even more crucial in 2DE electrophoresis where no universal method exists. The buffer typically contains a denaturing agent, urea or mixture of urea and thiourea, a non-ionic or zwitterionic detergent, a reducing agent to cleave disulfide-bonds, and ampholytes. The solubilization buffer is formulated to achieve optimal protein solubilization and prevent precipitation during the rehydration of the IPG strips-supported gel. Salts, ionic molecules and ionic detergents interfere with isoelectric focusing and compromise protein separation. The table below lists reagents commonly used for sample preparation. The concentration of each component needs to be determined empirically and experimental conditions require optimization. This can be achieved easily and rapidly using the ZOOM IPGRunner System.
| Component | Function | Concentration |
|---|---|---|
| Urea or Urea/Thiourea for membrane proteins | Protein denaturation and solubilization | 8 M or 9 M urea or 5–8 M urea with 2 M thiourea |
| Non-ionic detergent (CHAPS, CHAPSO, NP-40) | Protein solubilization and stabilization | 0.5–4% |
| DTT or DTE | Reduces disulfide bonds | 20 mM–100 mM |
| Ampholytes | Aid protein solubilization and maintain the pH gradient | 0.2–2% |
First dimension
The ZOOM IPGRunner System is an oil-free platform for isoelectric focusing of up to 12 samples in as little as 3 hours. The 7 cm ZOOM Strips are immobilized pH gradient (IPG) gel strips cast on a plastic support. They are available in broad and medium pH range formats (3–10, 4–6, and 6–10).
ZOOM IPG Strips options:
| Broad range: | Medium range: |
|---|---|
| pH 3–10 NonLinear | pH 6–10 |
| pH 3–10 Linear | pH 4–7 |
Wide pH range IPG strips allow the separation of most proteins in a single gel. The non-linear pH gradient improves the resolution of proteins in the middle of the pH range, where most proteins tend to focus, by expanding the pH range in the middle of the gel and compressing the pH at the acidic and basic ends of the gradient. By running IPG strips with overlapping pH range, the overall resolution of the separation is improved and more proteins can be analyzed, without a time-consuming sample fractionation step that can affect reproducibility.
The table below shows protein loading recommendation.
| IPG pH Range | Coomassie Staining | Silver Staining | Volume |
|---|---|---|---|
| Broad (3–10 L, NL) | 20–50 μg | 5–15 μg | 140 μl or 155 μl for overnight rehydration |
| Medium (6–10, 4–7) | 100–200 μg | 50–100 μg | 140 μl or 155 μl for overnight rehydration |
The ZOOM Carrier Ampholytes are small, soluble molecules with both positive and negative charge groups that sort at their relative position based upon their isoelectric field, setting up a pH gradient. Carrier ampholytes help to stabilize the pH gradient and current in IPG strips and aid in protein solubility, resulting in more reproducible IEF resolution.
Second dimension
Following isoelectric focusing, the ZOOM Strips are reduced, alkylated and equilibrated with NuPAGE LDS Sample Buffer. Equilibrated ZOOM IPG strips load easily into the NuPAGE Gels for 2D separation of proteins by molecular weight.
Protein gel staining
Following the SDS-PAGE, separated protein spots can be stained and visualized. The following protein stains combine high sensitivity with rapid and easy staining protocols and are compatible with downstream excision of the protein spot, tryptic-digestion and mass-spectrometry analysis.
| Protein Stain | Sensitivity | Staining time | Additional Information |
|---|---|---|---|
| SYPRO Ruby | 0.25–1 ng | 90 min microwave 18 hr standard | Fluorescent stain Ex: 280, 450nm Em: 610 nm |
| SilverQuest Silver Staining Kit | 0.3 ng | Std: 1 hr 30 min Microwave: 30 min | Includes ingredients for complete removal of silver ions |
| Simply Blue Safe Stain | > 7 ng | 12 min microwave 135 min standard | Single component– Ready to use |
| Pro-Q Emerald 488 Glycoprotein Gel and Blot Stain kit | 4 ng | ~6 hr | Ex: 510 nm Em: 520 nm |
| Pro-Q Emerald 300 Glycoprotein Gel and Blot Stain kit | 0.5 ng | ~5 hr | Ex: 280 nm Em: 530 nm Can also be detected with 300 nm UV illumination |
| Pro-Q Diamond Phosphoprotein Gel Staining Kit | 1-16 ng | 4–5 hr | Ex: 555 nm Em: 580 nm |
Protein spots can also be transferred to a membrane for immunodetection or for protein sequencing.
Welcome packs
Our first and second dimension welcome packs combine all the components necessary to get started with 2D electrophoresis. Just add the ZOOM IPG strips and carrier ampholytes that suit your needs.
See the table below for a complete list of the products included in each welcome pack:
| Name | Components | |
|---|---|---|
| First dimension | PowerEase™ Touch HV Power Supply and ZOOM™ IPGRunner™ Welcome Pack | PE Touch HV ZOOM IPGRunner Mini-Cell ZOOM IPGRunner Cassettes ZOOM Urea ZOOM CHAPS ZOOM Equilibration Trays |
| Second dimension | NuPAGE™ Bis-Tris XCell SureLock™ Mini-Cell Welcome Pack, 4 to 12%, IPG-Well | XCell SureLock Mini-Cell NuPAGE 4–12% Bis-Tris ZOOM gels NuPAGE LDS Sample Buffer (4X) NuPAGE Sample Reducing Agent (10X) NuPage MES Running Buffer (20X) PageRuler Plus Prestained Protein Ladder |
Ordering
First dimension
Second dimension
Protein gel staining
Documents
Sample preparation
Sample preparation is a fundamental step in any protein analysis workflow, but it is even more crucial in 2DE electrophoresis where no universal method exists. The buffer typically contains a denaturing agent, urea or mixture of urea and thiourea, a non-ionic or zwitterionic detergent, a reducing agent to cleave disulfide-bonds, and ampholytes. The solubilization buffer is formulated to achieve optimal protein solubilization and prevent precipitation during the rehydration of the IPG strips-supported gel. Salts, ionic molecules and ionic detergents interfere with isoelectric focusing and compromise protein separation. The table below lists reagents commonly used for sample preparation. The concentration of each component needs to be determined empirically and experimental conditions require optimization. This can be achieved easily and rapidly using the ZOOM IPGRunner System.
| Component | Function | Concentration |
|---|---|---|
| Urea or Urea/Thiourea for membrane proteins | Protein denaturation and solubilization | 8 M or 9 M urea or 5–8 M urea with 2 M thiourea |
| Non-ionic detergent (CHAPS, CHAPSO, NP-40) | Protein solubilization and stabilization | 0.5–4% |
| DTT or DTE | Reduces disulfide bonds | 20 mM–100 mM |
| Ampholytes | Aid protein solubilization and maintain the pH gradient | 0.2–2% |
First dimension
The ZOOM IPGRunner System is an oil-free platform for isoelectric focusing of up to 12 samples in as little as 3 hours. The 7 cm ZOOM Strips are immobilized pH gradient (IPG) gel strips cast on a plastic support. They are available in broad and medium pH range formats (3–10, 4–6, and 6–10).
ZOOM IPG Strips options:
| Broad range: | Medium range: |
|---|---|
| pH 3–10 NonLinear | pH 6–10 |
| pH 3–10 Linear | pH 4–7 |
Wide pH range IPG strips allow the separation of most proteins in a single gel. The non-linear pH gradient improves the resolution of proteins in the middle of the pH range, where most proteins tend to focus, by expanding the pH range in the middle of the gel and compressing the pH at the acidic and basic ends of the gradient. By running IPG strips with overlapping pH range, the overall resolution of the separation is improved and more proteins can be analyzed, without a time-consuming sample fractionation step that can affect reproducibility.
The table below shows protein loading recommendation.
| IPG pH Range | Coomassie Staining | Silver Staining | Volume |
|---|---|---|---|
| Broad (3–10 L, NL) | 20–50 μg | 5–15 μg | 140 μl or 155 μl for overnight rehydration |
| Medium (6–10, 4–7) | 100–200 μg | 50–100 μg | 140 μl or 155 μl for overnight rehydration |
The ZOOM Carrier Ampholytes are small, soluble molecules with both positive and negative charge groups that sort at their relative position based upon their isoelectric field, setting up a pH gradient. Carrier ampholytes help to stabilize the pH gradient and current in IPG strips and aid in protein solubility, resulting in more reproducible IEF resolution.
Second dimension
Following isoelectric focusing, the ZOOM Strips are reduced, alkylated and equilibrated with NuPAGE LDS Sample Buffer. Equilibrated ZOOM IPG strips load easily into the NuPAGE Gels for 2D separation of proteins by molecular weight.
Protein gel staining
Following the SDS-PAGE, separated protein spots can be stained and visualized. The following protein stains combine high sensitivity with rapid and easy staining protocols and are compatible with downstream excision of the protein spot, tryptic-digestion and mass-spectrometry analysis.
| Protein Stain | Sensitivity | Staining time | Additional Information |
|---|---|---|---|
| SYPRO Ruby | 0.25–1 ng | 90 min microwave 18 hr standard | Fluorescent stain Ex: 280, 450nm Em: 610 nm |
| SilverQuest Silver Staining Kit | 0.3 ng | Std: 1 hr 30 min Microwave: 30 min | Includes ingredients for complete removal of silver ions |
| Simply Blue Safe Stain | > 7 ng | 12 min microwave 135 min standard | Single component– Ready to use |
| Pro-Q Emerald 488 Glycoprotein Gel and Blot Stain kit | 4 ng | ~6 hr | Ex: 510 nm Em: 520 nm |
| Pro-Q Emerald 300 Glycoprotein Gel and Blot Stain kit | 0.5 ng | ~5 hr | Ex: 280 nm Em: 530 nm Can also be detected with 300 nm UV illumination |
| Pro-Q Diamond Phosphoprotein Gel Staining Kit | 1-16 ng | 4–5 hr | Ex: 555 nm Em: 580 nm |
Protein spots can also be transferred to a membrane for immunodetection or for protein sequencing.
Welcome packs
Our first and second dimension welcome packs combine all the components necessary to get started with 2D electrophoresis. Just add the ZOOM IPG strips and carrier ampholytes that suit your needs.
See the table below for a complete list of the products included in each welcome pack:
| Name | Components | |
|---|---|---|
| First dimension | PowerEase™ Touch HV Power Supply and ZOOM™ IPGRunner™ Welcome Pack | PE Touch HV ZOOM IPGRunner Mini-Cell ZOOM IPGRunner Cassettes ZOOM Urea ZOOM CHAPS ZOOM Equilibration Trays |
| Second dimension | NuPAGE™ Bis-Tris XCell SureLock™ Mini-Cell Welcome Pack, 4 to 12%, IPG-Well | XCell SureLock Mini-Cell NuPAGE 4–12% Bis-Tris ZOOM gels NuPAGE LDS Sample Buffer (4X) NuPAGE Sample Reducing Agent (10X) NuPage MES Running Buffer (20X) PageRuler Plus Prestained Protein Ladder |
Ordering
First dimension
Second dimension
Protein gel staining
Documents
References
- Boldt, A, Fortunato, D, Conti, A, Petersen, A, Ballmer-Weber, B, Lepp, U, Reese, G, and Becker, W-M “Analysis of the composition of an immunoglobulin E reactive high molecular weight protein complex of peanut extract containing Ara h 1 and Ara h 3/4” Proteomics 2005 Feb;5(3):675-86.
- Tetlow, IJ, Wait, R, Lu, Z, Akkasaeng, R, Bowsher, CG, Esposito, S, Kosar-Hashemi, B, Morell, MK, and Emes, MJ “Protein Phosphorylation in Amyloplasts Regulates Starch Branching Enzyme Activity and Protein-Protein Interactions” Plant Cell 2004 Mar;16:694-708.
- Zancai P, Dal Col J, Piccinin S, Guidoboni M, Cariati R, Rizzo S, Boiocchi M, Maestro R, Dolcetti R. “Retinoic acid stabilizes p27(Kip1) in EBV-immortalized lymphoblastoid B cell lines through enhanced proteasome-dependent degradation of the p45(Skp2) and Cks1 proteins” Oncogene. 2005 Feb 14;
- O’Farrell PH: High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975, 250: 4007-4021.
- Righetti, P. G. Isoelectric Focusing: Theory, Methodology and Applications. Elsevier Biomedical, Amsterdam. 1983
- Righetti, P. G. Immobilized pH Gradients: Theory and Methodology. Elsevier, Amsterdam. 1990
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For Research Use Only. Not for use in diagnostic procedures.