False labelling due to quenching failure of N-hydroxy-succinimide-ester-coupled dyes.
In comparative fluorescence gel electrophoresis experiments, cross-talk was detected. It was traced back to a failure in the quenching process in typical labelling protocols. Despite a huge excess of potential reaction sites for the N-hydroxy-succinimide-ester-coupled dye, sufficient active dye molecules were available after the quenching step to label protein molecules un-specifically. It could be shown that only a 100-fold increase in the amount of quencher will silence residual dye to such an extent that no artificial signals are detected.
Wang
Weiqun
W
Integrated Functional Genomics, Interdisciplinary Center for Clinical Research, Medical Faculty, University of Münster, Münster, Germany.
Ackermann
Doreen
D
Mehlich
Anna-Maria
AM
König
Simone
S
D002232:Carbocyanines / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004952:Esters / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008958:Models, Molecular; D040901:Proteomics; D013388:Succinimides / Q000737:chemistry*
D002232:Carbocyanines; D004952:Esters; D005456:Fluorescent Dyes; D013388:Succinimides