2-D Two Dimensional Gel Electrophoresis

Two dimensional gel electrophoresis (also called abbreviated as 2-D electrophoresis or 2-DE) is a method of gel electrophoresis which is commonly used to separate or analyze very similar in size, and also many proteins such as the complete set of proteins present in a given cell at once time, the proteome.

2-D gel electrophoresis separates the proteins in two dimensions, isoelectric point and mass. This allows the separation of proteins that otherwise could not be separated or distinguished on 1-D gels, including similarly sized proteins and many proteins (such as a proteome).

2-D Gel Table of Contents

• 2-D Gel Electrophoresis Background
• 2-D Gel Articles
• Separation by Isoelectric Point
• Two Dimensional Gel Electrophoresis Staining Methods
• 2-D Gel Electrophoresis Protocol
• Other 2-D Gel Electrophoresis Protocols
• Troubleshooting 2-D Gels
• 2-D Gel Electrophoresis Forum (You can ask any question about 2-D here! Also join the mailing list to receive up to the day questions and answers on 2-D gels from other science researchers and experts!)

Two Dimensional Gel Electrophoresis Background

Electrophoresis of all cellular proteins through an SDS-gel can separate proteins having relatively large differences in molecular weight however, it cannot readily resolve proteins having similar weights (e.g. a 53-kDA protein from a 52-kDA protein).

In the 1D gel electrophoresis method, the proteins are separated in one dimension (that is they are separated by mass only).

In 2-D electrophoresis, the separation begins with 1-D electrophoresis however there is a second separation that takes place, separating the proteins by charge in a direction 90 degrees from the first.

The result is that the analytes are spread out across a two dimensional surface rather than along a line. Thus, due to the fact that proteins are separated by not only mass but also by charge, it is unlikely that there will be more than one protein at one given spot on the 2-D gel. This is because it is unlikely that two proteins would share not only the same exact mass but also the same charge.

Therefore, on a 2-D gel, analytes are more effectively separated in 2-D electrophoresis than in 1-D electrophoresis due to the second separation step by charge.

The 2-D Gel Separation Method by Isoelectric Point

In order to separate proteins of a similar mass, another physical characteristic of proteins must be used. Another important physical protein property that may be used in separation is electrical charge, which is determined by the number of acidic and basic residues in a protein and their group charge.

Two unrelated proteins having similar masses are unlikely to have identical net charges because their amino acid sequence would be different, and thus the charge and number of acidic and basic residues would give a different net charge. This would allow these proteins to be separated by 2-D, but not by 1-D gel electrophoresis.

In the 2-D gel separation method, proteins are actually separated by isoelectric point (isoelectric point (pI) is the pH at which a particular molecule or surface carries no net electrical charge.).

A pH gradient is applied to a gel and an electric potential is applied across the gel, making one end more positive than the other. At all pHs aother than their isoelectric point, proteins will be charged. If they are positively charged, they will be pulled towards the more negative end of the gel and if they are negatively charged they will be pulled to the more positive end of the gel. Once they reach the region of the gel with pH corresponding to their isoelectric point, however, they will become neutraly charged and remain in that spot.

2-D Gel Electrophoresis Staining Methods

Protein Detection Protocols for Two Dimensional Electrophoresis

After separation, the proteins are separated out on the 2-D gel, however are invisible to the naked eye. To visualize the proteins on the gel, very sensitive staining and detection methods of the proteins must be carried out.

This is due to several facts:

1. The gel lanes can only accept a certain volume of cell lysate or protein solution.
2. Although the lysate/solution can be precipitated and concentrated to load more protein, usually less protein is loaded on the 2D gel as excessive protein amounts make it difficult to analyze the gel due to large spot sizes.
3. The separation of a large amount of protein (mgs), or even thousands of different proteins from one small lane, into a relatively large two dimensionsional gel generates spots of proteins that are very low abudance / concentration (ng or picograms!) that are impossible or difficult to detect by ordinary stains or protein detection methods.
4. A low abundance / low copy number protein can be separated, but may not even be detected due to the fact that the most sensitive stains only have a detection limit of about a 1/2 a nanogram. Picogram spot levels are currently very difficult or impossible to detect with staining.

In 2D electrophoresis, very sensitive stains are therefore required. These proteins can then be detected by a variety of means, but the most common is silver staining and coomasie stain.

Silver Staining 2D Gels

In 2-D gel silver staining, a silver colloid is applied to the gel. The silver bonds to cysteine groups within the protein. The silver is then darkened by exposure to UV light. The darkness of the silver in the spot, can be related to the amount of silver and therefore the amount of protein at a given location on the gel. This measurement can only give approximate amounts, but is adequate for most purposes.

Coomasie Staining of 2D Gels

Coomassie dye binds to proteins via physisorption to amino acids such as the aromatic amino acids, arginine, and histidine. Coomasie is also used in the Bradford Method. Sensitive non-toxic coomasie stains have been generated which allow visualization of spots containing less than 1 ng of protein.

SYPRO Staining of 2D Gels

SYPRO RUBY is a very sensitive 2D protein detection method stain, allowing visualization of about 1/2 a ng of protein at a given spot. The disadvantage is the method requires UV light to visualize the stain, thus requiring the usage of a UV light. This makes cutting of the spots difficult and potentially hazardous due to possible UV exposure.

2-D Gel Electrophoresis Protocol

A two dimensional gel electrophoresis protocol for 7 cm IPG strips Amersham GE Sensing.

2D Gel Sample Preparation

• Protein samples usually need to be desalted or dialyzed. Dialysis can be conducted with large dialysis bags for large volumes, or the mini slide-a-lyzer (Pierce) for small volumes.
• Dialysis was conducted with 1 L of 0.3mM ammonium bicarbonate over 30 minutes using the slide-a-lyzer dialysis kits (Pierce).
• After dialysis, samples were lyophilized using a lyophilizer for 1 ½ hours.
• Lyophilized samples were then reconstituted with 400 l rehydration buffer.

IEF IsoElectric Focusing

Step 1. Rehydration of IPG Strips

• The drystrip rehydration isoelectrofocusing tray was carefully cleaned with IPGphor cleaning solution (Amersham GE sensing).
• Make sure the tray is dry not wet, as sample will migrate along the water channels.
• Pipette 125 l onto the circular area of the tray.
• Using tweezers, carefully remove IPG strips from plastic protector.
• Some IPG strips have a plastic coversheet or strip protecting the gel side. Remove this using forceps / tweezers before using these strips.
• Gently lay down the strip gel side down onto the sample.
• Remove any bubbles.
• Lay down on the end of the tray lane Mineral Oil and make sure you push it down the lane until it completely covers the IPG strip. Make sure the mineral oil overlays the whole strip as this prevents evaporation during the rehydration steps.