Validation of self-made fluorescent cyanine dyes for 2D gel-based multi-fluorescence protein analysis.
Two-dimensional gel electrophoresis (2-DE) in combination with quantitative multi-fluorescence protein analysis (MFA) is the most versatile methodical tool for differential analysis of protein mixtures or even complex proteomes. It is based on covalent labelling of proteins with fluorescent cyanine dyes (Ethyl-Cy2, Propyl-Cy3 or Methyl-Cy5) before isoelectric focussing, enabling differential tagging of up to three samples which are finally separated on the same 2-D gel. To minimize costs and to increase the number of possible experiments, the cyanine dye NHS esters were synthesized in our own lab according to a protocol published by Jung and Kim (2006) . Self-made cyanine dyes were tested by studying their labelling and fluorescent properties and possible effects on the electrophoretic mobility of labelled proteins. To validate the potential use as labels in 2-DE/MFA experiments, dyes were used for the differential analysis of the proteome of thrombin-stimulated human vascular smooth muscle cells (VSMCs).
Kaber
Gernot
G
Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum Düsseldorf, Universitätsstrasse 1, Düsseldorf, Germany. kaber@uni-duesseldorf.de
Vormbrock
Ingo
I
Hartwig
Sonja
S
Lehr
Stefan
S
Schrör
Karsten
K
D000818:Animals; D002232:Carbocyanines / Q000032:analysis / Q000138:chemical synthesis*; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional / Q000191:economics / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000138:chemical synthesis*; D006801:Humans; D008099:Liver / Q000737:chemistry; D051379:Mice; D032389:Myocytes, Smooth Muscle / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry*; D020543:Proteome / Q000737:chemistry*; D013917:Thrombin / Q000494:pharmacology; D014680:Veins / Q000166:cytology / Q000378:metabolism
D002232:Carbocyanines; D005456:Fluorescent Dyes; D011506:Proteins; D020543:Proteome; D013917:Thrombin