Two dimensional gel electrophoresis

Two dimensional gel electrophoresis, commonly abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. In 1-D electrophoresis, proteins (or other analytes) are separated in one dimension, so that all the analytes will lie along a line but be separated from each other by some property. 2-D electrophoresis begins with 1-D electrophoresis but then separates the analytes by a second property in a direction 90 degrees from the first. The result is that the analytes are spread out across a 2-D surface rather than along a line. Because it is less likely that two analytes will be the same in both properties than that they will be the same in just one property, analytes are more effectively separated in 2-D electrophoresis than in 1-D electrophoresis.

The two dimensions that proteins are separated into in this technique correspond to isoelectric point and mass.

To separate the proteins by isoelectric point, a gradient of pH 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.

Before separating the proteins by mass, they are treated with sodium dodecyl sulfate (SDS). This denatures the proteins (that is, it unfolds them into long, straight molecules) and attaches attaches a number of SDS molecules roughly proportional to the protein's length. Because a protein's length (when unfolded) is roughly proportional to its mass, this is equivalent to saying that it attaches a number of SDS molecules roughly proportional to the protein's mass. Since the SDS molecules are negatively charged, the result of this is that all of the proteins will have approximately the same mass-to-charge ratio as each other. Next, an electric potential is again applied, but at a 90 degree angle from the first field. The proteins will be attracted to the more negative side of the gel proportionally to their mass-to-charge ratio. As previously explained, this ratio will be nearly the same for all proteins. The proteins' progress will be slowed by frictional forces. This frictional slowing is roughly inversely proportional to the protein's size which, as noted previously, is roughly proportional to its mass when the protein is denatured. The electric feild is applied for as long as it takes the smallest protein to reach the far end of the gel.

The result of this is a gel with proteins spread out on its surface. These proteins can then be detected by a variety of means, but the most common is silver staining. In this case, a silver colloid is applied to the gel. The silver bonds to cysteine groups within the protein. The silver is darkened by exposure to ultra-violet light. The darkness of the silver 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.

See also

• electrophoresis

External links

• A 2-D electrophoresis tutorial on the web site of the Parasitology Group at Aberystwyth University
• Discussion forum about 2D gel image analysis