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Introduction to 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) using Ettan™ DIGE System- Detect an increased number of real differences in protein expression.
- Identify the smallest possible expression differences.
- Guarantee statistical confidence.
- Eliminate gel-to-gel variation.
Workflow for Ettan DIGE system showing co-separation of three fluorescently labeled samples separated on a single 2-D gel. One sample is an internal standard.
Ettan™ DIGE (2-D Fluorescence Difference Gel Electrophoresis) system enables the prelabeling and separation of up to three samples on a single 2-D gel providing accurate quantitation, reproducibility, ease of use, and increased throughput. Reduced system variability enables accurate study of protein abundance differences against a baseline of inherent biological variation.
The system consists of three main components:
- Protein visualization¾CyDye™ DIGE fluors.
- Image capture¾Typhoon™ Variable Mode Imager or Ettan DIGE Imager.
- Image analysis¾DeCyder™ 2-D Differential Analysis Software or ImageMaster 2D Platinum DIGE Enabled
software.
Ettan DIGE system brings statistical confidence and reliability to 2-D electrophoresis. Combining novel proprietary technologies in fluorescence multiplexing and image analysis, Ettan DIGE system is a fully integrated system offering significant benefits over classical 2-D electrophoresis. Ettan DIGE system uses a defined procedure that includes an internal standard to generate the most accurate results. Using the system, up to three protein samples are labeled with novel size- and charge-matched CyDye DIGE fluors and co-separated on the same 2-D electrophoresis gel.
Imaging is performed using the Typhoon Variable Mode Imager or Ettan DIGE Imager. Image analysis is provided by DeCyder 2-D Differential Analysis Software, with or without DeCyder Extended Data Analysis Software module, or with ImageMaster™ 2D Platinum v6.0 DIGE Enabled software.
Internal standard and experimental design
The major source of error in a 2-D experiment is gel-to-gel variation. To eliminate this, an internal standard is run on all gels within an experiment. Multiplexing using Ettan DIGE system is the only easy way in which an internal standard can be used.
Labeling and internal standardization for real 2-D differences
Using Ettan DIGE system, up to three protein samples labeled with different CyDye DIGE fluors can be run on the same 2-D gel simultaneously. One of these, the internal standard, results from the pooling of aliquots of all biological samples in the experiment. Two sets of dyes are available, the minimal dyes and the saturation dyes from the scarce sample labeling kit. With the minimal dyes, the internal standard is labeled with one CyDye DIGE Fluor minimal dye (e.g., Cy™2) and is run together with individual samples labeled with other CyDye DIGE Fluor minimal dyes (e.g., Cy3 and Cy5). With the saturation dyes from the CyDye DIGE Fluor Labeling Kit for Scarce Samples, the internal standard is labeled with one dye (e.g., Cy3) and is run together with individual samples labeled with the Cy5 dye. This means that every protein from all samples will be represented in the internal standard and each protein can therefore be compared to
itself within the internal standard to generate a ratio of relative abundance.
The same internal standard is run on all gels within an experimental series thereby creating an intrinsic link across all gels. Normalization of the internal standard across gels allows the ratio of relative abundance of the same protein across gels to be compared directly, separating gel-to-gel variation from biological variation. Even small differences in expression levels can then be determined by comparing the ratio obtained from one fluorescently labeled sample directly with another. As a result, it is possible to see 10% differences in protein abundance between samples, with greater than 95% statistical confidence.
Experimental design for CyDye DIGE Fluor minimal dyes
An example of a recommended experimental design to derive statistical data on any differences between control and treated samples labeled with CyDye DIGE Fluor minimal dyes Cy2, Cy3, or Cy5 is outlined below. Six control and six treated samples are examined, each sample having its own internal standard according to the table (a pool is created by adding equal amounts of each sample together).
| Gel number | Cy™2 | Cy™3 | Cy™5 |
| 1 | Pooled standard | Control 1 | Treated 3 |
| 2 | Pooled standard | Control 2 | Treated 2 |
| 3 | Pooled standard | Control 3 | Treated 1 |
| 4 | Pooled standard | Treated 6 | Control 4 |
| 5 | Pooled standard | Treated 5 | Control 5 |
| 6 | Pooled standard | Treated 4 | Control 6 |
Experimental variation is very low with Ettan DIGE system and we therefore recommend biological replicates and not replicates of the same sample. If the same experiment is carried out using conventional 2-D electrophoresis using one sample per gel, at least 12 gels would be required. To gain any meaningful statistical data using this approach would require a minimum of three replicates of each, making 36 gels in total.
Experimental design for CyDye DIGE Fluor saturation dyes from the Scarce Sample Labeling Kit
An example of a recommended experimental design to derive statistical data on any differences between control and treated samples labeled with CyDye™ DIGE Fluor saturation dyes Cy3 or Cy5 is outlined here. Six control and six treated samples are examined, each sample having its own internal standard.
| Gel number | Cy™3 | Cy™5 |
| 1 | Pooled standard | Control 1 |
| 2 | Pooled standard | Control 2 |
| 3 | Pooled standard | Control 3 |
| 4 | Pooled standard | Control 4 |
| 5 | Pooled standard | Control 5 |
| 6 | Pooled standard | Control 6 |
| 7 | Pooled standard | Treated 1 |
| 8 | Pooled standard | Treated 2 |
| 9 | Pooled standard | Treated 3 |
| 10 | Pooled standard | Treated 4 |
| 11 | Pooled standard | Treated 5 |
| 12 | Pooled standard | Treated 6 |
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