Abstract
Reverse phase protein array (RPPA) techniques allow the quantitative analysis of signal transduction events in a high-throughput format. Sensitivity is important for RPPA-based detection approaches, since numerous signaling proteins or posttranslational modifications are present at low levels. Especially, the proteomic analysis of clinical samples exposes its own challenges with respect to sensitivity. Antibody-mediated signal amplification (AMSA) is a novel strategy relying on sequential incubation steps with fluorescently labeled secondary antibodies reactive against each other. AMSA is a simple extension of the standard quantification in the near-infrared range and is highly specific and robust. In this chapter, we present the amplification protocol and application examples for the time-resolved analysis of signaling pathways as well as protein profiling of clinical samples.
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Acknowledgments
We thank Maike Wosch and Annika Bittmann for their excellent technical assistance. This work was supported by the German Federal Ministry for Education and Science in the framework of the Program for Medical Genome Research (grants 01GS0890 and 01GS0864), the Program for Medical Systems Biology (grant 0315396B), as well as the Helmholtz Systems Biology Initiative (SBCancer).
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Brase, J.C., Mannsperger, H., Sültmann, H., Korf, U. (2011). Antibody-Mediated Signal Amplification for Reverse Phase Protein Array-Based Protein Quantification. In: Korf, U. (eds) Protein Microarrays. Methods in Molecular Biology, vol 785. Humana Press. https://doi.org/10.1007/978-1-61779-286-1_5
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DOI: https://doi.org/10.1007/978-1-61779-286-1_5
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