Ettan DIGE System: Raising the Standard of 2-D
EttanTM DIGE System comprises a defined procedure for 2-D that uses an internal standard to generate the most accurate results.
The 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 pooled standard should be run on all gels within an experiment. Multiplexing is the only way in which an internal standard can be used.
Labelling and internal standardization for real differences
With Ettan DIGE System, up to three labeled protein samples 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. This internal standard is labelled with one CyDye™ DIGE Fluor minimal dyes (e.g. Cy™2) and is run together with individual samples, experimental and control, labelled with other CyDye DIGE Fluor minimal dyes (e.g. Cy3 or Cy5). 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 expression.
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 expression 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 be determined by comparing the ratio obtained from one fluorescent-labeled sample directly with another. As a result, it is possible to see < 10% differences in protein expression between samples, with > 95% statistical confidence. | 
