title: Impact of quenching failure of Cy dyes in differential gel electrophoresis.
abstract: BACKGROUND
  Differential gel electrophoresis (DIGE) is a technology widely used for protein expression analysis. It is based on labelling with fluorescent Cy dyes. In comparative fluorescence gel electrophoresis experiments, however, unspecific labelling using N-hydroxy-succinimide-ester-based labelling protocols was recently detected. Cross-talk was observed due to failure of the quenching process. Here, the impact of this effect for DIGE experiments was investigated.
  
  
  RESULTS
  Experiments to test quenching efficiency were performed in replicate using Escherichia coli lysate. Parameters such as the amount of dye and quencher were varied. Labelling and quenching were reversed in one experiment. Differences in protein spot volumes due to limited quenching were determined. For some spots twice the volume was detected underscoring the importance of proper control of silencing of active dye.
  
  
  CONCLUSIONS
  It could be demonstrated that uncontrolled labelling increased protein spot volume, even doubling it in some cases. Moreover, proteins responded differently to the protocol. Such unpredictable and unspecific processes are not acceptable in protein regulation studies so that it is necessary to validate the correct amount of quencher for individual samples before the DIGE experiment is performed. Increase of the concentration of lysine, which is used as quencher, from 10 mM to 2500 mM, was sufficient to silence the dye. Alternatively, active dye molecules can be removed by filtration.
authors.affiliation: Integrated Functional Genomics, Core Unit of the Interdisciplinary Center for Clinical Research, Medical Faculty, University of Münster, Münster, Germany.
authors.forename: Weiqun
authors.initials: W
authors.lastname: Wang
authors.forename: Doreen
authors.initials: D
authors.lastname: Ackermann
authors.forename: Anna-Maria
authors.initials: AM
authors.lastname: Mehlich
authors.forename: Simone
authors.initials: S
authors.lastname: König
mesh_terms: D004396:Coloring Agents / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D013194:Staining and Labeling
chemical_list: D004396:Coloring Agents