Chapter title |
Solubility Characterization and Imaging of Intrabodies Using GFP-Fusions
|
---|---|
Chapter number | 9 |
Book title |
Synthetic Antibodies
|
Published in |
Methods in molecular biology, March 2017
|
DOI | 10.1007/978-1-4939-6857-2_9 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6855-8, 978-1-4939-6857-2
|
Authors |
Emilie Rebaud, Pierre Martineau, Laurence Guglielmi, Emilie Renaud |
Editors |
Thomas Tiller |
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. |
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