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Cell-Free Assays

The Reductionist Approach to the Study of NADPH Oxidase Assembly, or “All You Wanted to Know About Cell-Free Assays but Did Not Dare to Ask”

  • Protocol
Book cover Neutrophil Methods and Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 412))

Abstract

The superoxide (O2 •−)-generating enzyme complex of phagocytes, known as the NADPH oxidase, can be assayed in a number of in vitro cell-free (or broken cell) systems. These consist of a mixture of the individual components of the NADPH oxidase, derived from resting phagocytes or in the form of purified recombinant proteins, exposed to an activating agent (or situation), in the presence of NADPH and oxygen. O2 •− produced by the mixture is measured by being trapped immediately after its generation with an appropriate acceptor in a kinetic assay, which permits the calculation of the linear rate of O2 •− production over time. Cell-free assays are distinguished from whole-cell assays or assays performed on membranes derived from stimulated cells by the fact that all components in the reaction are derived from resting, nonstimulated cells and, thus, the steps of NADPH oxidase activation (precatalytic [assembly] and catalytic) occur in vitro. Cell-free assays played a paramount role in the identification of the components of the NADPH oxidase complex, the diagnosis of various forms of chronic granulomatous disease (CGD), and, more recently, the analysis of the domains present on the components of the NADPH oxidase participating in protein-protein interactions leading to the assembly of the active complex.

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Authors and Affiliations

  1. The Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research and the Ela Kodesz Institute of Host Defense against Infectious Diseases, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

    Ariel Mizrahi, Yelena Ugolev, Iris Dahan, Yevgeny Berdichevsky & Edgar Pick

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  1. Shahar Molshanski-Mor
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  2. Ariel Mizrahi
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  3. Yelena Ugolev
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  4. Iris Dahan
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  5. Yevgeny Berdichevsky
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  6. Edgar Pick
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Editors and Affiliations

  1. Deparment of Veterinary Molecular Biology, Montana State University, Bozeman, MT

    Mark T. Quinn

  2. Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT

    Frank R. DeLeo

  3. Departments of Immunology and Cell Biology, The Scripps Institute, La Jolla, CA

    Gary M. Bokoch

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Molshanski-Mor, S., Mizrahi, A., Ugolev, Y., Dahan, I., Berdichevsky, Y., Pick, E. (2007). Cell-Free Assays. In: Quinn, M.T., DeLeo, F.R., Bokoch, G.M. (eds) Neutrophil Methods and Protocols. Methods in Molecular Biology™, vol 412. Humana Press. https://doi.org/10.1007/978-1-59745-467-4_25

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  • DOI: https://doi.org/10.1007/978-1-59745-467-4_25

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