title: Detecting HIV-1 Tat in Cell Culture Supernatants by ELISA or Western Blot.
abstract: HIV-1 Tat is efficiently secreted by HIV-1-infected or Tat-transfected cells. Accordingly, Tat concentrations in the nanomolar range have been measured in the sera of HIV-1-infected patients, and this protein acts as a viral toxin on bystander cells. Nevertheless, assaying Tat concentration in media or sera is not that straightforward because extracellular Tat is unstable and particularly sensitive to oxidation. Moreover, most anti-Tat antibodies display limited affinity. Here, we describe methods to quantify extracellular Tat using a sandwich ELISA or Western blotting when Tat is secreted by suspension or adherent cells, respectively. In both cases it is important to capture exported Tat using antibodies before any Tat oxidation occurs; otherwise it will become denatured and unreactive toward antibodies.
authors.affiliation: CPBS, UMR 5236 CNRS, University of Montpellier, 1919 Route de Mende, 34923, Montpellier Cedex 05, France.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Rayne
authors.affiliation: CPBS, UMR 5236 CNRS, University of Montpellier, 1919 Route de Mende, 34923, Montpellier Cedex 05, France.
authors.forename: Solène
authors.initials: S
authors.lastname: Debaisieux
authors.affiliation: Institut des Neurosciences Cellulaires et Intégratives, CNRS UPR3212, University of Strasbourg, Strasbourg, France.
authors.forename: Annie
authors.initials: A
authors.lastname: Tu
authors.affiliation: CPBS, UMR 5236 CNRS, University of Montpellier, 1919 Route de Mende, 34923, Montpellier Cedex 05, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Chopard
authors.affiliation: Institut des Neurosciences Cellulaires et Intégratives, CNRS UPR3212, University of Strasbourg, Strasbourg, France.
authors.forename: Petra
authors.initials: P
authors.lastname: Tryoen-Toth
authors.affiliation: CPBS, UMR 5236 CNRS, University of Montpellier, 1919 Route de Mende, 34923, Montpellier Cedex 05, France. bruno.beaumelle@cpbs.cnrs.fr.
authors.forename: Bruno
authors.initials: B
authors.lastname: Beaumelle
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000302:isolation & purification*; D006801:Humans; D019169:Jurkat Cells; D051381:Rats; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000032:analysis*
chemical_list: D054322:tat Gene Products, Human Immunodeficiency Virus
keywords: ELISA; HIV-1; Immunoblots; Secretion; Tat