Azure Biosystems
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 (Figure 1) 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.
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 offers several benefits, including higher sensitivity, accuracy, and reproducibility, enabling better protein detection. Additionally, compared to standard techniques, 2D DIGE requires fewer gels, making it a more cost-efficient choice.
2D DIGE is more sensitive than standard 2D gel electrophoresis. Overall, utilizing 2D DIGE can improve the overall quality and efficiency of protein analysis, providing researchers with a better understanding of their samples.
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:
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.
Nowadays, there are advanced laser-scanning systems on the market capable of multiple applications, including 2D DIGE, and more. The new Azure Sapphire FL Biomolecular Imager (Figure 2) is ideal for imaging 2D DIGE experiments. It offers customizable and user-changeable optical modules, providing many potential combinations of fluorescent tags and the ability to compare two or more samples on one 2D gel.
Using a reliable analysis software is also important for the analysis of 2D DIGE experiments. SameSpots 2D Analysis Software from Azure provides image QC to identify problems such as spot saturation and carries out spot identification and intensity comparisons. This software can also account for variations in gel migration to carry out alignment of single-channel 2D gels run at different times. Contact us today for a demo of the Sapphire FL using SameSpots.
Able to compare two or more samples on one 2D gel
Check out this publication that uses the 2D DIGE capability from the Sapphire to scan protein gels extracted using trichloroacetic acid/acetone precipitation in Type 2 diabetes research.
Alshahrani et al. from Al Faisal University and King Saud University evaluated proteomic changes in plasma from patients who were obese, obese with type-2 diabetes, or obese and diabetic while taking metformin. The 2D gels were scanned by the Sapphire.
Looking for a full list of applications?
In recent work, Ekhzaimy et al1 took advantage of the three-channel fluorescence imaging capability of the Sapphire Biomolecular Imager to conduct a proteomics study of liraglutide treatment
