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Robust Protein Profiling with Complex Antibody Microarrays in a Dual-Colour Mode

  • Christoph Schröder
  • Mohamed S. S. Alhamdani
  • Kurt Fellenberg
  • Andrea Bauer
  • Anette Jacob
  • Jörg D. Hoheisel
Part of the Methods in Molecular Biology book series (MIMB, volume 785)

Abstract

Antibody microarrays are a multiplexing technique for the analyses of hundreds of different analytes in parallel from small sample volumes of few microlitres only. With sensitivities in the picomolar to femtomolar range, they are gaining importance in proteomic analyses. These sensitivities can be obtained for complex protein samples without any pre-fractionation or signal amplification. Also, no expensive or elaborate protein depletion steps are needed. As with custom DNA-microarrays, the implementation of a dual-colour assay adds to assay robustness and reproducibility and was therefore a focus of our technical implementation. In order to perform antibody microarray experiments for large sets of samples and analytes in a robust manner, it was essential to optimise the experimental layout, the protein extraction, labelling and incubation as well as data processing steps. Here, we present our current protocol, which is used for the simultaneous analysis of the abundance of more than 800 proteins in plasma, urine, and tissue samples.

Key words

Antibody microarray Proteomic profiling Dual-colour analysis, direct sample labelling Plasma profiling Multiplexed immunoassay 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Christoph Schröder
    • 1
  • Mohamed S. S. Alhamdani
    • 1
  • Kurt Fellenberg
    • 2
  • Andrea Bauer
    • 1
  • Anette Jacob
    • 1
  • Jörg D. Hoheisel
    • 1
  1. 1.Functional Genome AnalysisGerman Cancer Research Center (DKFZ)HeidelbergGermany
  2. 2.Chair of Proteomics and BioanalyticsTechnical University MunichFreisingGermany

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