Two-Dimensional Difference Gel Electrophoresis

What is 2D DIGE?

Two-dimensional gel electrophoresis (2D DIGE) facilitates the comparison of proteomes (all proteins in a sample) in order to identify differences in specific protein levels. In 2D DIGE, two or more samples are run on a single gel. Before electrophoresis, the proteins in each sample are labeled with different fluorescent tags. Then the resulting 2D gel is imaged under excitation and emission conditions optimal for each fluorophore, generating separate 2D gel images for each sample. The images can be directly compared since no correction must be made for differences in migration due to variations in gel consistency or running conditions.

The power of proteomics

2D gel electrophoresis is a core tool in the study of proteomics. Traditional polyacrylamide gel electrophoresis (PAGE) of proteins separates proteins by size; however, 2D PAGE adds an additional step wherein the proteins are first separated by isoelectric point.

In the resulting 2D distribution, up to 10,000 proteins can be differentiated, providing an overview of the sample’s proteome. Comparing proteomes between biological samples has powerful scientific potential to, for example, identify biomarkers associated with disease states in serum samples, identify differences between cancerous and noncancerous samples of a specific tissue, or identify changes associated with response to therapy.

Data analysis using 2D DIGE

2D DIGE experiments involve normalizing each image, identifying the protein spots, matching spots that represent the same protein across different samples, quantifying the intensity of each spot, and calculating the ratio of intensity for each spot between the samples being compared. Differences in the intensity between spots representing the same proteins indicate a change in abundance of the protein between samples.

2D DIGE experiment including three samples of E. coli bacterial lysate labeled with Cy2, Cy3, and Cy5 dyes
Figure 1. 2D DIGE experiment including three samples of E. coli bacterial lysate labeled with Cy2, Cy3, and Cy5 dyes. The 2D gel parameters included isoelectric focusing over a broad range (pH 3.5 to 10) followed by separation by size on a 12.5% SDS-PAGE gel. The gel was imaged using the Azure Sapphire Biomolecular Imager in the 488, 520, and 658 channels with a pixel size of 100 m and a focal depth of 0.67 mm. The image shown is an overlay of the images captured in each of the three channels (blue, red, and green).

Why image quality is important for 2D DIGE

The quality of 2D DIGE data depends on the quality of the fluorescent images of the 2D gel. Accurate identification and definition of protein spots and accurate quantitation of the intensity of each protein spot require:

  • images that have a wide dynamic range
  • images in which no spot intensities are saturated
  • images of high sensitivity to detect low-abundance proteins
  • images free from position effects across the field of view

Imagers capable of 2D DIGE analysis

The Azure Sapphire Biomolecular Imager is ideal for imaging 2D DIGE experiments. The Sapphire offers up to four fluorescent channels providing many potential combinations of fluorescent tags and the ability to compare two or more samples on one 2D gel.

Azure Biosystems offers SameSpots 2D Analysis Software to facilitate the analysis of 2D DIGE experiments. SameSpots provides image QC to identify problems such as spot saturation and carries out spot identification and intensity comparisons. SameSpots can also account for variations in gel migration to carry out alignment of single-channel 2D gels run at different times.

Related Products

Able to compare two or more samples on one 2D gel

Check out this 2D DIGE publication that uses the Sapphire to scan protein gels extracted using trichloroacetic acid/acetone precipitation in Type 2 diabetes research.

Looking for a full list of applications?

Documents

Document TypeDescription
Application NoteMeasuring Histamine Levels in Food SamplesDOWNLOAD
Application NotePerforming an ELISA with the Ao Absorbance Microplate ReaderDOWNLOAD

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