Antibody-mediated signal amplification for reverse phase protein array-based protein quantification.

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.