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Reverse Phase Protein Microarrays: Fluorometric and Colorimetric Detection

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Protein Microarray for Disease Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 723))

Abstract

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.

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Author information

Authors and Affiliations

  1. George Mason University, Manassas, VA, USA

    Rosa I. Gallagher, Alessandra Silvestri, Emanuel F. Petricoin III & Lance A. Liotta

  2. CRO-IRCCS, National Cancer Institute, Aviano, Italy

    Alessandra Silvestri

  3. Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA

    Virginia Espina

Authors
  1. Rosa I. Gallagher
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  2. Alessandra Silvestri
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  3. Emanuel F. Petricoin III
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  4. Lance A. Liotta
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  5. Virginia Espina
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Corresponding author

Correspondence to Virginia Espina .

Editor information

Editors and Affiliations

  1. Cancer Vaccine Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Avenue Louis Pasteur 77, Boston, 02115, Massachusetts, USA

    Catherine J. Wu

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Gallagher, R.I., Silvestri, A., Petricoin, E.F., Liotta, L.A., Espina, V. (2011). Reverse Phase Protein Microarrays: Fluorometric and Colorimetric Detection. In: Wu, C. (eds) Protein Microarray for Disease Analysis. Methods in Molecular Biology, vol 723. Humana Press. https://doi.org/10.1007/978-1-61779-043-0_18

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  • DOI: https://doi.org/10.1007/978-1-61779-043-0_18

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-042-3

  • Online ISBN: 978-1-61779-043-0

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