Biotechnology of Plant Secondary Metabolism pp 99-107

Part of the Methods in Molecular Biology book series (MIMB, volume 1405) | Cite as

A Western Blot Protocol for Detection of Proteins Heterologously Expressed in Xenopus laevis Oocytes

  • Morten Egevang Jørgensen
  • Hussam Hassan Nour-Eldin
  • Barbara Ann Halkier
Protocol

Abstract

Oocytes of the African clawed frog, Xenopus laevis, are often used for expression and biochemical characterization of transporter proteins as the oocytes are particularly suitable for uptake assays and electrophysiological recordings. Assessment of the expression level of expressed transporters at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins heterologously expressed in Xenopus oocytes.

Key words

Transport protein quantification Single oocyte Western blot Xenopus oocytes 

References

  1. 1.
    Boorer KJ, Forde BG, Leigh RA, Miller AJ (1992) Functional expression of a plant plasma membrane transporter in Xenopus oocytes. FEBS Lett 302:166–168CrossRefPubMedGoogle Scholar
  2. 2.
    Theodoulou FL, Miller AJ (1995) Xenopus oocytes as a heterologous expression system for plant proteins. Mol Biotechnol 3:101–115CrossRefPubMedGoogle Scholar
  3. 3.
    Sumikawa K, Houghton M, Emtage JS, Richards BM, Barnard EA (1981) Active multi-subunit ACh receptor assembled by translation of heterologous mRNA in Xenopus oocytes. Nature 292:862–864CrossRefPubMedGoogle Scholar
  4. 4.
    Sigel E (1990) Use of Xenopus oocytes for the functional expression of plasma membrane proteins. J Membr Biol 117:201–221CrossRefPubMedGoogle Scholar
  5. 5.
    Calamita G, Bishai WR, Preston GM, Guggino WB, Agre P (1995) Molecular cloning and characterization of AqpZ, a water channel from Escherichia coli. J Biol Chem 270:29063–29066CrossRefPubMedGoogle Scholar
  6. 6.
    Wahl R, Wippel K, Goos S, Kämper J, Sauer N (2010) A novel high-affinity sucrose transporter is required for virulence of the plant pathogen Ustilago maydis. PLoS Biol 8:e1000303PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Redshaw MR, Follett BK (1971) The crystalline yolk-platelet proteins and their soluble plasma precursor in an amphibian, Xenopus laevis. Biochem J 124:759–766PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Nour-Eldin HH, Norholm MHH, Halkier BA (2006) Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes. Plant Methods 2:17PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Krieg PA, Melton DA (1984) Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res 12:7057–7070PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Galili G, Altschuler Y, Ceriotti A (1995) Synthesis of plant proteins in heterologous systems: Xenopus laevis oocytes. In: David W, Galbraith DPB, Hans JB (eds) Method cell biol. Academic, San Diego, pp 497–517Google Scholar
  11. 11.
    Liu XS, Liu XJ (2006) Oocyte isolation and enucleation. Methods Mol Biol 322:31–41CrossRefPubMedGoogle Scholar
  12. 12.
    Wilson IA, Niman HL, Houghten RA, Cherenson AR, Connolly ML, Lerner RA (1984) The structure of an antigenic determinant in a protein. Cell 37:767–778CrossRefPubMedGoogle Scholar
  13. 13.
    Gassmann M, Grenacher B, Rohde B, Vogel J (2009) Quantifying Western blots: pitfalls of densitometry. Electrophoresis 30:1845–1855CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Morten Egevang Jørgensen
    • 1
  • Hussam Hassan Nour-Eldin
    • 1
  • Barbara Ann Halkier
    • 1
  1. 1.DynaMo Center, Department of Plant and Environmental Sciences, Faculty of ScienceUniversity of CopenhagenFrederiksberg CDenmark

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