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Solubility Characterization and Imaging of Intrabodies Using GFP-Fusions

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1575)

Abstract

Ectopically expressed intracellular recombinant antibodies, or intrabodies, are powerful tools to visualize proteins and study their function in fixed or living cells. However, many intrabodies are insoluble and aggregate in the reducing environment of the cytosol. To solve this problem, we describe an approach based on GFP-tagged intrabodies. In this protocol, the GFP is used both as a folding-reporter to select correctly folded intrabodies and as a fluorescent tag to localize the scFv and its associated antigen in eukaryotic cells. Starting from a scFv gene cloned in a retroviral vector, we describe retrovirus production, cell line transduction, and soluble intrabody characterization by microscopy and FACS analysis.

Key words

Intrabody scFv GFP Folding Aggregation Degradation 

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.IRCM, Institut de Recherche en Cancérologie de MontpellierMontpellierFrance
  2. 2.INSERM, U1194MontpellierFrance
  3. 3.Université de MontpellierMontpellierFrance
  4. 4.Institut régional du Cancer de MontpellierMontpellierFrance

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