Summary
We describe procedures for intracellular expression of scFv in eukaryotic cells. Starting from a scFv gene cloned in a phage-display vector, we describe the cloning step into a mammalian expression vector, the transient transfection of a HeLa cell line, and the monitoring of intrabody expression by immunofluorescence staining and FACS analysis.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Lobato, M. N., and Rabbitts, T. H. (2003) Intracellular antibodies and challenges facing their use as therapeutic agents. Trends Mol. Med. 9, 390–396.
Cardinale, A., Lener, M., Messina, S., Cattaneo, A., and Biocca, S. (1998) The mode of action of Y13-259 scFv fragment intracellularly expressed in mammalian cells. FEBS Lett. 439, 197–202.
Lener, M., Horn, I. R., Cardinale, A., Messina, S., Nielsen, U. B., Rybak, S. M., Hoogenboom, H. R., Cattaneo, A., and Biocca, S. (2000) Diverting a protein from its cellular location by intracellular antibodies. The case of p21Ras. Eur. J. Biochem. 267, 1196–1205.
Wörn, A., and Plückthun A. (2001) Stability engineering of antibody single-chain Fv fragments. J. Mol. Biol. 305, 989–1010.
Auf der Maur, A., Zahnd, C., Fischer, F., Spinelli, S., Honegger, A., Cambillau, C., Escher, D., Pluckthun, A., and Barberis, A. (2002) Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework. J. Biol. Chem. 277, 45075–45085.
Tanaka, T., Chung, G. T. Y., Forster, A., Lobato, M. N., and Rabbitts, T. H. (2003) De novo production of diverse intracellular antibody libraries. Nucleic Acids Res. 31, e23.
Villani, M. E., Carli, M. D., Donini, M., Traversini, G., Lico, C., Franconi, R., Benvenuto, E., and Desiderio, A. (2008) Validation of a stable recombinant antibodies repertoire for the direct selection of functional intracellular reagents. J. Immunol. Methods 329, 11–20.
Philibert, P., Stoessel, A., Wang, W., Sibler, A., Bec, N., Larroque, C., Saven, J. G., Courtete, J., Weiss, E., and Martineau, P. (2007) A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm. BMC Biotechnol. 7, 81.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Guglielmi, L., Martineau, P. (2009). Intrabody Expression in Eukaryotic Cells. In: Aitken, R. (eds) Antibody Phage Display. Methods in Molecular Biology, vol 562. Humana Press. https://doi.org/10.1007/978-1-60327-302-2_15
Download citation
DOI: https://doi.org/10.1007/978-1-60327-302-2_15
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-301-5
Online ISBN: 978-1-60327-302-2
eBook Packages: Springer Protocols