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Optimization of immunoprecipitation–western blot analysis in detecting GW182-associated components of GW/P bodies

Nature Protocols volume 4, pages 674–685 (2009)Cite this article

Abstract

Characterizing the components of GW/processing bodies is key to elucidating RNA interference and messenger RNA processing pathways. This protocol addresses challenges in isolating a low-abundance protein GW182 and GW body (GWB)-associated proteins by building on previous reports that used polyclonal sera containing autoantibodies to GW/P body components. This protocol uses commercially available monoclonal antibodies to GW182 that are covalently coupled to Protein A or G sepharose beads and then used to immunoprecipitate GW182 and associated proteins from cell extracts. Immunoprecipitates are separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and probed by western blot with antibodies directed to proteins of interest. This protocol, which is expected to take 4–5 d, provides a biochemical approach for detecting GW182 and associated proteins in biological samples and thus facilitates the elucidation of the diverse functions of GWBs. It is expected that this protocol can be adapted to the detection of other RNA-binding complexes.

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Figure 1: Summary of the steps involved and the approximate time required in detecting GW182 and associated proteins that are components of GWBs.
Figure 2: Antibodies to GW182-immunoprecipitated Ago2, Dicer and XRN1 proteins from HeLa cell extracts as examined by WB analysis.
Figure 3: Example of successful covalent coupling of antibodies to sepharose beads.
Figure 4: Example of a Ponceau S-stained nitrocellulose membrane containing 40 μg of protein per lane from a HeLa cell lysate extract.

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Acknowledgements

This work was supported in part by the Canadian Institutes for Health Research Grant MOP-57674 and NIH Grant AI47859. M.J.F. holds the Arthritis Society Chair. J.J.M. is supported by a CIHR Doctoral Research Award in the Area of Clinical Research and by an Alberta Heritage Foundation for Medical Research Studentship Award.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada

    Joanna J Moser & Marvin J Fritzler

  2. Department of Oral Biology, University of Florida College of Dentistry, Health Science Centre, Gainesville, Florida, USA

    Edward K L Chan

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Correspondence to Marvin J Fritzler.

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Moser, J., Chan, E. & Fritzler, M. Optimization of immunoprecipitation–western blot analysis in detecting GW182-associated components of GW/P bodies. Nat Protoc 4, 674–685 (2009). https://doi.org/10.1038/nprot.2009.34

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