Accuracy & Reproducibility

2D DIGE platform is highly accurate and reproducible, eliminating the need of running technical replicates, which translates into high quality data at a lower cost. The following examples assessed the effects of different CyDye, gel-to-gel variations and samples on the data output.

Example-1: Effects of different fluorescent dyes

The same liver lysate was labeled separately with Cy3 and Cy5, then resolved on the same 2D DIGE gel. All protein spots matched perfectly in the overlay image, indicating high accuracy and reproducibility regardless of fluorescent dyes.

Cy3: Liver Lysate

Cy3 labeled mouse liver proteome image

Cy5: Liver Lysate

Cy5 labeled mouse liver proteome image

Cy3 / Cy5 – Overlay

Overlay image of Cy3 and Cy5 labeled mouse liver proteome

Example 2: Effects of in-gel and cross-gel 2D DIGE

The same cell lysate sample was labeled separately with Cy2, Cy3 and Cy5, then resolved on the same 2D DIGE gel (Gel-1). The same experiment was repeated and same set of data was collected (Gel-2). The data showed high reproducibility within the gel and across different gels.

DeCyder Software Analysis

Cy3 / Cy2Cy3 / Cy5Cy2 / Cy5
Within Gel-1
2 S.D: after filter
Total spot number
Included spot number
Excluded spot number

1.22873
2235
915
1320

1.17503
2210
1070
1140

1.22577
2210
933
1277
Within Gel-2
2 S.D: after filter
Total spot number
Included spot number
Excluded spot number

1.21323
2256
1026
1230

1.18648
2250
1051
1199

1.22116
2246
1095
1151
Across Gels
2 S.D after filter

1.29214

1.25791

1.31146

Example 3: Effects of freeze/thaw

The extracted cell lysate sample in 2D lysis buffer is stored at – 80oC. The same cell lysate sample was frozen/thaw a few times, then labeled with different CyDye and resolved on 2D DIGE. The data showed nearly identical protein profiling of these samples.

Cy2 labeled fresh cell lysate proteome image
Cy3 labeled 1-thaw cell lysate proteome image
Cy5 labeled 3-thaw cell lysate proteome image
Triple color overlay image of Cy2, Cy3 and Cy5 labeled cell lysate proteome image

Example 4: Reproducibility of paired samples

The same pair of mouse liver proteins were run on 2 different 2D DIGE gels. The 2 overlay images showed that the unchanged (yellow) and changed (red and green) spots remained the same between the 2 gels, suggesting that 2D DIGE data is highly reproducible.

WT / S33A (pair 1)

Overlay image of paired mouse liver proteome from Gel-1

WT / S33A (pair 2)

Overlay image of paired mouse liver proteome from Gel-2