Unit

UNIT 27.7 Preparation and Use of Reverse Protein Microarrays

  1. Elisa Pin1,2,
  2. Giulia Federici1,3,
  3. Emanuel F. Petricoin III1

Published Online: 3 FEB 2014

DOI: 10.1002/0471140864.ps2707s75

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Pin, E., Federici, G. and Petricoin, E. F. 2014. Preparation and Use of Reverse Protein Microarrays. Current Protocols in Protein Science. 75:27.7:27.7.1–27.7.29.

Author Information

  1. 1

    Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia

  2. 2

    Division of Experimental Oncology 2, Centro di Riferimento Oncologico-IRCCS, National Cancer Institute, Aviano, Italy

  3. 3

    Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità (ISS), Rome, Italy

Publication History

  1. Published Online: 3 FEB 2014

Abstract

Reverse-phase protein array (RPPA) is a multiplex, high-throughput proteomic technique for profiling the activation status of signal transduction pathways involved in cancer survival and progression, potentially allowing for identification of new biomarkers and drug targets. On RPPA, the entire patient proteome is immobilized on a spot and single proteins can be quantified across a set of samples, spotted on the same array, with high specificity and sensitivity. Array immunostaining and signal amplification systems are used to generate a signal proportional to the concentration of the analyte. Dedicated scanners and software are used to detect spots, measure intensity, subtract background, normalize signal, and generate a numeric value as output. The generated output file is then analyzed using several different bioinformatic and biostatistical tools. In this unit, the RPPA procedure is described in depth, from sample handling and preparation to data analysis, with particular emphasis on tissue sample analysis. Curr. Protoc. Protein Sci. 75:27.7.1-27.7.29. © 2014 by John Wiley & Sons, Inc.

Keywords:

  • tissues;
  • cells;
  • protein;
  • microarray;
  • antibody;
  • analysis