Difference gel electrophoresis (DIGE) is a form of
gel electrophoresis
Gel electrophoresis is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...
where up to three different
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
samples can be labeled with size-matched, charge-matched spectrally resolvable
fluorescent dye
A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...
s (for example Cy3, Cy5, Cy2) prior to
two dimensional gel electrophoresis
Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first ...
.
[Unlü M, Morgan ME, Minden JS. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis. 1997 Oct;18(11):2071-7. PMI]
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Procedure
The three samples are mixed and loaded onto IEF (isolectric focusing chromatography) for first dimension and the strip is transferred to a SDS PAGE. After the gel electrophoresis, the gel is scanned with the
excitation wavelength
Absorption spectroscopy refers to spectroscopic techniques that measure the absorption of radiation, as a function of frequency or wavelength, due to its interaction with a sample. The sample absorbs energy, i.e., photons, from the radiating f ...
of each dye one after the other, so each sample can be seen separately (if we scan the gel at the
excitation wavelength
Absorption spectroscopy refers to spectroscopic techniques that measure the absorption of radiation, as a function of frequency or wavelength, due to its interaction with a sample. The sample absorbs energy, i.e., photons, from the radiating f ...
of the Cy3 dye, we will see in the gel only the sample that was labeled with that dye). This technique is used to see changes in protein abundance (for example, between a sample of a healthy person and a sample of a person with disease), 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 (change in isoelectric point), vertical (change in size) or diagonal (change in both size and isoelectric point). 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 (e.g. healthy/diseased, virulent/non-virulent) 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.
See also
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PROTOMAP
References
{{Electrophoresis
Electrophoresis