Fluorescent dual colour 2D-protein gel electrophoresis for rapid detection of differences in protein pattern with standard image analysis software.
The use of two different fluorescent dyes in two-dimensional (2D) polyacrylamide gel electrophoresis was recently described and termed difference gel electrophoresis (DIGE). Thereby differences between protein samples could be accomplished by fluorescently tagging the samples with different dyes as well as co-separation and visualisation in a single gel. We adapted this method to the ampholyte technique, using newly available fluorescent dyes and three common image software systems for analysis. Working with protein lysates from tumour cell lines with defined added proteins we found that the technique is reproducible, sensitive and fast, because it circumvents the necessity of matching several 2D gels. This is mainly due to the fact that the generated images from the two different fluorescent channels could be superimposed by standard image analysis, so that changes in the protein pattern could be easily detected either by a different colour or by comparing grey values of corresponding spots. This method will be especially helpful in comparing proteins from normal and tumour tissue to highlight changes in genesis and progression in cancer.
Von Eggeling
F
F
Institut fur Humangenetik und Anthropologie, Kollegiengasse 10, D-07740 Jena, Germany. fegg@mti-n.uni-jena.de
Gawriljuk
A
A
Fiedler
W
W
Ernst
G
G
Claussen
U
U
Klose
J
J
Römer
I
I
D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007091:Image Processing, Computer-Assisted; D011506:Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity; D012984:Software; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D011506:Proteins