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Reverse-phase protein lysate microarrays for cell signaling analysis

Nature Protocols volume 3, pages 1796–1808 (2008)Cite this article

Abstract

'Reverse-phase' protein lysate microarray (RPA) assays use micro-scale, cell lysate dot blots that are printed to a substrate, followed by quantitative immunochemical protein detection, known to be particularly effective across many samples. Large-scale sample collection is a labor-intensive and time-consuming process; the information yielded from RPA assays, however, provides unique opportunities to experimentally interpret theoretical protein networks quantitatively. When specific antibodies are used, RPA can generate 1,000 times more data points using 10,000 times less sample volume than an ordinary western blot, enabling researchers to monitor quantitative proteomic responses for various time-scale and input-dose gradients simultaneously. Hence, the RPA system can be an excellent method for experimental validation of theoretical protein network models. Besides the initial screening of primary antibodies, collection of several hundreds of sample lysates from 1- to 8-h periods can be completed in ∼10 d; subsequent RPA printing and signal detection steps require an additional 2–3 d.

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Figure 1: Outline of reverse-phase protein lysate microarray protocol for quantitative protein expression analysis.
Figure 2: 'Reverse-phase' protein lysate microarray (RPA) images and data output.
Figure 3: Screenshot of the Aushon BioSystems 2470 Microarray Graphical User Interface (GUI).
Figure 4: Preparation of twofold serial dilutions in a 384-and 1,536-well microtiter plate.

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Acknowledgements

We thank Drs. John Austin, Toni Holway and Peter Honkanen of Auslon BioSystems for excellent protein microarray technical support, and Dr. Lynn Young for customizing image processing programs.

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Authors and Affiliations

  1. Molecular Translational Technology Section, Molecular Therapeutics Program, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, 20892, Maryland, USA

    Brett Spurrier & Satoshi Nishizuka

  2. Aushon BioSystems, 43 Manning Rd, Billerica, 01821, Massachusetts, USA

    Brett Spurrier

  3. University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, 27599, North Carolina, USA

    Sundhar Ramalingam

  4. Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., NCI-Frederick, Frederick, 21702, Maryland, USA

    Satoshi Nishizuka

  5. Department of Surgery, Molecular Therapeutics Laboratory, Iwate Medical University, Uchimaru 19-1, Morioka, 020-8505, Iwate, Japan

    Satoshi Nishizuka

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  1. Brett Spurrier
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Correspondence to Satoshi Nishizuka.

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Spurrier, B., Ramalingam, S. & Nishizuka, S. Reverse-phase protein lysate microarrays for cell signaling analysis. Nat Protoc 3, 1796–1808 (2008). https://doi.org/10.1038/nprot.2008.179

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