Impact of Quenching Failure of Cy Dyes in Differential Gel Electrophoresis

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 label...

Authors: Schneider-Wang, Weiqun
Ackermann, Doreen
Mehlich, Anna-Maria
König, Simone
Division/Institute:FB 05: Medizinische Fakultät
Document types:Article
Media types:Text
Publication date:2011
Date of publication on miami:20.02.2013
Modification date:16.04.2019
Edition statement:[Electronic ed.]
Source:PLoS ONE 6 (2011) 3, e18098
DDC Subject:610: Medizin und Gesundheit
License:CC BY 2.5
Language:English
Notes:Finanziert durch den Open-Access-Publikationsfonds 2011/2012 der Deutschen Forschungsgemeinschaft (DFG) und der Westfälischen Wilhelms-Universität Münster (WWU Münster).
Format:PDF document
URN:urn:nbn:de:hbz:6-87379485034
Permalink:http://nbn-resolving.de/urn:nbn:de:hbz:6-87379485034
Other Identifiers:DOI: doi:10.1371/journal.pone.0018098
Digital documents:journal.pone.0018098.pdf

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. Methodology/Principal Findings: 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/Significance: 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.