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Generation and purification of highly specific antibodies for detecting post-translationally modified proteins in vivo

Nature Protocols volume 9, pages 375–395 (2014)Cite this article

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Abstract

Post-translational modifications alter protein structure, affecting activity, stability, localization and/or binding partners. Antibodies that specifically recognize post-translationally modified proteins have a number of uses including immunocytochemistry and immunoprecipitation of the modified protein to purify protein-protein and protein-nucleic acid complexes. However, antibodies directed at modified sites on individual proteins are often nonspecific. Here we describe a protocol to purify polyclonal antibodies that specifically detect the modified protein of interest. The approach uses iterative rounds of subtraction and affinity purification, using stringent washes to remove antibodies that recognize the unmodified protein and low sequence complexity epitopes containing the modified amino acid. Dot blot and western blot assays are used to assess antibody preparation specificity. The approach is designed to overcome the common occurrence that a single round of subtraction and affinity purification is not sufficient to obtain a modified protein-specific antibody preparation. One full round of antibody purification and specificity testing takes 6 d of discontinuous time.

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Figure 1: Spatial control of NOS-3 phosphorylation in the C. elegans germ-line tissue.
Figure 2: Workflow for phospho-antibody purification.
Figure 3: Removal of nonspecific antibodies from the phospho-CGH-1 preparation through multiple rounds of affinity purification as assessed by western blots of adult C. elegans lysates.
Figure 4: Testing the efficiency of peptide conjugation to activated BSA.
Figure 5: Dot blot to test specificity toward the CGH-1 phosphopeptides and non-phosphopeptides.
Figure 6: Genetic tests for specificity of the purified anti-pCGH-1 polyclonal antibody preparation.
Figure 7: Dot blotting to identify fractions containing the antipeptide antibody.
Figure 8: Staining for pCGH-1 and activated MPK-1 ERK overlap in the germ-line tissue.
Figure 9: Phosphorylation of CGH-1 alters protein subcellular localization.

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Acknowledgements

Work in the T.S. laboratory on this project was supported by National Science Foundation grant no. 0416502 and US National Institutes of Health (NIH) grant no. GM085150. Work in S.A.'s laboratory on this project was supported by NIH grant no. GM98200, an Institutional Research Grant from a Cancer Center Supplemental Grant (CCSG) to The University of Texas M.D. Anderson Cancer Center and the Center for Genetics and Genomics, The University of Texas M.D. Anderson Cancer Center. We thank A. Golden (NIH/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)), D. Greenstein (University of Minnesota) and C. Spike (University of Minnesota) for extensive comments on the manuscript.

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  1. Department of Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA

    Swathi Arur

  2. Department of Genetics, Washington University in St. Louis, St. Louis, Missouri, USA

    Tim Schedl

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S.A. and T.S. designed, implemented and wrote the protocol.

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Arur, S., Schedl, T. Generation and purification of highly specific antibodies for detecting post-translationally modified proteins in vivo. Nat Protoc 9, 375–395 (2014). https://doi.org/10.1038/nprot.2014.017

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