Difference gel electrophoresis


Difference gel electrophoresis is a form of gel electrophoresis where up to three different protein samples can be labeled with size-matched, charge-matched spectrally resolvable fluorescent dyes prior to two dimensional gel electrophoresis.

Procedure

The three samples are mixed and loaded onto IEF for first dimension and the strip is transferred to a SDS PAGE. After the gel electrophoresis, the gel is scanned with the excitation wavelength of each dye one after the other, so each sample can be seen separately. This technique is used to see changes in protein abundance, post-translational modifications, truncations and any modification that might change the size or isoelectric point of proteins. The binary shifts might be left to right, vertical or diagonal. Reciprocal Labeling is done to make sure the changes seen are not due to dye-dependent interactions.

Advantages

It overcomes limitations in traditional 2D electrophoresis that are due to inter-gel variation. This can be considerable even with identical samples. Since the proteins from the different sample types are run on the same gel they can be directly compared. To do this with traditional 2D electrophoresis requires large numbers of time-consuming repeats.

Standards

In experiments comprising several gels, a common technique is to include an internal standard in each gel. The internal standard is prepared by mixing together several or all of the samples in the experiment. This allows the measurement of the abundance of a protein in each sample relative to the internal standard. Since the amounts of each protein in the internal standard is known to be the same in every gel, this method reduces inter-gel variation.electrophoresis incorporating a pooled internal standard.