title: Solubility Characterization and Imaging of Intrabodies Using GFP-Fusions.
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.
authors.affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, 34298, France.|INSERM, U1194, Campus Val d'Aurelle, Montpellier, 34298, France.|Université de Montpellier, Montpellier, 34090, France.|Institut régional du Cancer de Montpellier, Montpellier, 34298, France.
authors.forename: Emilie
authors.initials: E
authors.lastname: Renaud
authors.affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, 34298, France. pierre.martineau@inserm.fr.|INSERM, U1194, Campus Val d'Aurelle, Montpellier, 34298, France. pierre.martineau@inserm.fr.|Université de Montpellier, Montpellier, 34090, France. pierre.martineau@inserm.fr.|Institut régional du Cancer de Montpellier, Montpellier, 34298, France. pierre.martineau@inserm.fr.
authors.forename: Pierre
authors.initials: P
authors.lastname: Martineau
authors.affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, 34298, France. laurence.guglielmi@inserm.fr.|INSERM, U1194, Campus Val d'Aurelle, Montpellier, 34298, France. laurence.guglielmi@inserm.fr.|Université de Montpellier, Montpellier, 34090, France. laurence.guglielmi@inserm.fr.|Institut régional du Cancer de Montpellier, Montpellier, 34298, France. laurence.guglielmi@inserm.fr.
authors.forename: Laurence
authors.initials: L
authors.lastname: Guglielmi
mesh_terms: D002469:Cell Separation; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D061986:MCF-7 Cells; D008853:Microscopy; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics / Q000502:physiology; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000378:metabolism; D012995:Solubility; D014161:Transduction, Genetic
chemical_list: D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D049452:Green Fluorescent Proteins
keywords: Aggregation; Degradation; Folding; GFP; Intrabody; scFv