Reverse Phase Protein Microarrays: Fluorometric and Colorimetric Detection

Rosa I. Gallagher, Alessandra Silvestri, Emanuel F. Petricoin, Lance A. Liotta, Virginia Espina*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

19 Scopus citations


The Reverse Phase Protein Microarray (RPMA) is an array platform used to quantitate proteins and their posttranslationally modified forms. RPMAs are applicable for profiling key cellular signaling pathways and protein networks, allowing direct comparison of the activation state of proteins from multiple samples within the same array. The RPMA format consists of proteins immobilized directly on a nitrocellulose substratum. The analyte is subsequently probed with a primary antibody and a series of reagents for signal amplification and detection. Due to the diversity, low concentration, and large dynamic range of protein analytes, RPMAs require stringent signal amplification methods, high quality image acquisition, and software capable of precisely analyzing spot intensities on an array. Microarray detection strategies can be either fluorescent or colorimetric. The choice of a detection system depends on (a) the expected analyte concentration, (b) type of microarray imaging system, and (c) type of sample. The focus of this chapter is to describe RPMA detection and imaging using fluorescent and colorimetric (diaminobenzidine (DAB)) methods.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages27
StatePublished - 2011
Externally publishedYes

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Colorimetric
  • Detection
  • Fluorometric
  • Image acquisition
  • Immunostaining
  • Microarray analysis
  • Protein
  • Reverse phase protein microarray


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