Advertisement

Immunoprecipitation of Membrane Proteins from Arabidopsis thaliana Root Tissue

  • Sascha Waidmann
  • Lucinda De-Araujo
  • Jürgen Kleine-Vehn
  • Barbara Korbei
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1761)

Abstract

Here, we present different methods for immunoprecipitating membrane proteins of Arabidopsis thaliana root material. We describe two extraction methods for the precipitation either for an integral membrane protein of the endoplasmic reticulum (ER) or a peripheral membrane protein partially localized at the plasma membrane, where we precipitate the protein out of the total membrane as well as total cytosolic fractions.

Key words

Immunoprecipitation Integral membrane proteins Peripheral membrane proteins Membrane fraction Cytosolic fraction Cross-linked beads Arabidopsis thaliana 

Notes

Acknowledgments

Establishment for this protocol was supported by a grant from the Austrian science fund FWF (P 29754 to SW, JK-V; V382 to BK), the Austrian Academy of Science to (LD-A (DOC-ÖAW 24195), the Hochschuljubiläumsstiftung der Stadt Wien (H-235701/2014 to BK), the Vienna Science and Technology Fund (WWTF) (to JK-V), and the European Research Council (ERC) (Starting Grant 639478-AuxinER to JK-V).

References

  1. 1.
    Avila JR, Lee JS, Torii KU (2015) Co-Immunoprecipitation of membrane-bound receptors. Arabidopsis Book 13:e0180CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Barbez E, Kubes M, Rolcik J, Beziat C, Pencik A, Wang B, Rosquete MR, Zhu J, Dobrev PI, Lee Y, Zazimalova E, Petrasek J, Geisler M, Friml J, Kleine-Vehn J (2012) A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants. Nature 485:119–122CrossRefPubMedGoogle Scholar
  3. 3.
    Korbei B, Moulinier-Anzola J, De-Araujo L, Lucyshyn D, Retzer K, Khan MA, Luschnig C (2013) Arabidopsis TOL proteins act as gatekeepers for vacuolar sorting of PIN2 plasma membrane protein. Curr Biol 23:2500–2505CrossRefPubMedGoogle Scholar
  4. 4.
    Labeta MO, Fernandez N, Festenstein H (1988) Solubilisation effect of Nonidet P-40, triton X-100 and CHAPS in the detection of MHC-like glycoproteins. J Immunol Methods 112:133–138CrossRefPubMedGoogle Scholar
  5. 5.
    Leitner J, Luschnig C (2014) Ubiquitylation-mediated control of polar auxin transport: analysis of Arabidopsis PIN2 auxin transport protein. Methods Mol Biol 1209:233–249CrossRefPubMedGoogle Scholar
  6. 6.
    Wendrich JR, Boeren S, Moller BK, Weijers D, De Rybel B (2017) In vivo identification of plant protein complexes using IP-MS/MS. Methods Mol Biol 1497:147–158CrossRefPubMedGoogle Scholar
  7. 7.
    Abas L, Luschnig C (2010) Maximum yields of microsomal-type membranes from small amounts of plant material without requiring ultracentrifugation. Anal Biochem 401:217–227CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Berleth ES, Pickart CM (1996) Mechanism of ubiquitin conjugating enzyme E2-230K: catalysis involving a thiol relay? Biochemistry 35:1664–1671CrossRefPubMedGoogle Scholar
  9. 9.
    Haltiwanger RS, Grove K, Philipsberg GA (1998) Modulation of O-linked N-acetylglucosamine levels on nuclear and cytoplasmic proteins in vivo using the peptide O-GlcNAc-beta-N-acetylglucosaminidase inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate. J Biol Chem 273:3611–3617CrossRefPubMedGoogle Scholar
  10. 10.
    Sousa MM, Steen KW, Hagen L, Slupphaug G (2011) Antibody cross-linking and target elution protocols used for immunoprecipitation significantly modulate signal-to noise ratio in downstream 2D-PAGE analysis. Proteome Sci 9:45CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Sascha Waidmann
    • 1
  • Lucinda De-Araujo
    • 1
  • Jürgen Kleine-Vehn
    • 1
  • Barbara Korbei
    • 1
  1. 1.Department of Applied Genetics and Cell Biology (DAGZ)University of Natural Resources and Life Sciences (BOKU)ViennaAustria

Personalised recommendations