2D DIGE Proteomics Service

2D-DIGE proteomics analysis, which labels different protein samples with up to three fluorescent tags, offers significant advantages over conventional 2D-PAGE and other proteomics platforms:

  • Higher sensitivity: Fluorescent dyes have a sensitivity of 0.2 ng/spot, versus the sensitivity of coomassie at 100 ng/spot or silver staining at 1 ng/spot
  • Higher accuracy: The extremely high spot resolution enables accurate spot quantitation. Differences in protein expression as small as 10% can be detected
  • Higher reproducibility: Nearly identical data was obtained on the same sample labeled with different CyDye on the same gel or cross different gels, thus eliminating the need of running technical replicates
  • Broader spectrum: Each gel can resolve ~5000 protein spots, allowing a wide dynamic range detection/quantitation of low abundant proteins, large proteins and small peptides
  • Fewer number of gels: Each gel can accommodate up to 3 different samples
  • Lower cost: The price is based on the number of gels, not the number of samples. Price covers from sample preparation to publication-ready data, and complimentary consultation with your project leader
  • Fast turn-around: 1 week

Main applications:

2D DIGE experimental procedure

2D DIGE & mass spectrometry full procedure
  1. Sample preparation: Proteins are extracted from cells or tissues of interest. Protein concentrations are determined by protein assay and adjusted to the desired concentration.
  2. Sample labeling with fluorescent dyes: Equal amount of protein extract was labeled by fluorescent dyes (size and charge matched), and the spectrally resolvable dyes enable simultaneous co-separation and analysis of up to 3 samples on a single multiplexed gel.
  3. 2D gel electrophoresis: Up to 3 samples can be simultaneously separated on a single 2D gel, with isoelectric focusing (IEF) in the 1st dimension and SDS polyacrylamide gel electrophoresis (SDS-PAGE) in the 2nd dimension.
  4. Image acquisition: After electrophoresis, the gel is scanned using a Typhoon image scanner. Each scan reveals one of the CyDye signals (Cy2, Cy3 and Cy5).
  5. Image analysis: ImageQuant software is used to generate the image presentation data including the single and overlay images.
  6. Quantitative analysis: Comparative analysis of all spots using DeCyder “in-gel” or “cross-gel” analysis software. The protein expression ratios between different samples or different groups of samples will be generated.
  7. Automated spot picking: Following the spot picking design using DeCyder software, protein spots of interest can be automatically picked from the 2D gel with the Ettan Spot Picker, followed by Protein Identification by Mass Spectrometry.
  8. Cluster Analysis: A complimentary service for projects with 50 or more identified proteins.
  9. Validation: We offer 2D Western Blot services to validate the 2D DIGE data.

2D DIGE Images

Table of Contents
SalivaPlant
UrineC. elegans
E. coliZebrafish
YeastMouse & Rat Brain
DrosophilaRat Tear

Saliva

2D images of human saliva proteome

Saliva pH 4-7

2D DIGE image of human saliva proteome proteome (pH4-7)

Saliva pH 3-10

2D DIGE image of human saliva proteome proteome (pH3-10)

Urine

2D images of human adult urine proteome

pH 4-7

2D DIGE image of human urine proteome pH4-7

pH 3-10

2D DIGE image of human urine proteome pH3-10

E. coli

2D images of E. coli Proteome (pH 3-10 NL)

2D DIGE image of E. coli proteome (pH3-10 NL)
2D DIGE image of E. coli Proteome (pH3-10 NL) in black/white

Yeast

2D image of yeast (pH 3-10 NL)

2D DIGE image of yeast proteome (pH3-10 NL)
2D DIGE image of yeast proteome (pH3-10 NL) in black/white

Drosophila

2D image of drosophila proteome (pH 3-10 NL)

2D DIGE image of drosophila proteome (pH3-10 NL)

Plant

2D image of plant (N. benthamiana) proteome (pH 4-7)

2D image of plant (N. benthamiana) proteome (pH4-7)

C. elegans

2D images of C. elegans proteome

C. elegans: pH 4-7

2D DIGE image of Caenorhabditis elegans proteome (pH4-7)

C. elegans: pH 3-10

2D DIGE image of Caenorhabditis elegans proteome (pH3-10)

Zebrafish

2D image of zebrafish proteome (pH 3-10L)

2D DIGE image of zebrafish proteome

Mouse & Rat Brain

2D images of mouse & rat brain proteome

Mouse Brain: pH 3-10

2D DIGE Image of Mouse Brain Proteome (pH3-10)

Rat Brain: pH 3-10

2D DIGE Image of Rat Brain Proteome (pH3-10)

Rat Tear

2D image of rat tear proteome

2D DIGE Image of Rat Tear Proteome

2D DIGE FAQs

Frequently asked questions about 2D-DIGE advantages, process, time & cost:

Compared to 2D-gel, 2D-DIGE offers higher sensitivity, higher accuracy, fewer number of gels, faster turn around, and a lower price.

Learn the difference

The sensitivity of 2D DIGE is about 0.2 ng/spot.

2D DIGE is very accurate due to the extremely high spot resolution. Differences in protein expression as small as 10% can be detected.

Highly reproducible: identical data can be obtained on the same sample labeled with different CyDye on the same gel or cross different gels (see an example).

No

2000-5000 protein spots using pH3-10 IPG strip. If using additional IPG strips such as pH 4-7, pH 6-9 and pH 7-11, over 5000 spots can be detected.

Most customers request to start the experiment with an Analytical Gel for the following reasons:

  1. Analytical gels use about 25 µg of each sample while prep-gels use about 250 µg of each sample. The total protein load for analytical gel is about 75 µg, while total protein load for prep-gels is about 750 µg. Since Analytical Gel contains lower sample amount resulting in less ion and salt in IEF, it will provide more accurate quantitation of protein changes) and much higher image quality for publication/presentation purposes.
  2. After running the analytical gel, the in-gel data analysis will be done. Based on the analytical gel results, you can put more control or treatment samples if your desired spots are from control (usually down-regulation), or from treatment (usually up-regulation or newly induced proteins). In contrast, Prep-gel is much less likely to provide such information due to the excess amount of protein.
  3. From the analytical gel, you will know if you need to focus on a specific region (i.e. with most changes) in the next 2-D experiment by changing the pH range for IEF or the percentage of the SDS-gel (for resolving lower or higher MW protein spots). In contrast, Prep-gel is much less likely to provide such information due to the excess amount of protein.
  4. The main purpose of running Prep-gel is to obtain enough protein amount for the protein ID by Mass Spectrometry.

5-7 days for Analytical gel.

7-9 days for Prep-gel, spot picking and protein identification by mass spectrometry.

The data report contains two main parts:

  • ImageQuant analysis: gel images of individual samples and overlay of two sample images
  • DeCyder analysis: quantitative analysis and comparison of all spots between different samples.

Yes, we can send you the raw data generated by instrument software free of charge within 1 year of the completion of project. If you request raw data after 1 year, it may incur a charge based on the actual time to retrieve them from achieve.

Let’s assume a new customer from academics who is interested in finding the differential protein expression among 2-3 samples, followed by obtaining Protein ID for the top 10 spots. Please note that the prices are per gels, not per sample. Each gel can load up to 3 samples. Following would be the estimated cost:

Service Description2 samples3 samples
2D DIGE Analytical Gel$1050*$1195*
2D DIGE Preparative Gel$1200$1295
Spot Picking (up to 96 spots per gel)$250$250
10 Protein ID by Mass Spectrometry$1250$1250
Total cost$3750$3990

* Only applies for new customers who submit standard protein samples with protein concentration >5mg/ml. The discount is subject to approval from Finance. The discounted price applies for the first 2D DIGE Analytical gel. The discounted price does NOT apply for Preparative gels.