Detecting HIV-1 Tat in Cell Culture Supernatants by ELISA or Western Blot.
Methods Mol Biol
; 1354: 329-42, 2016.
Article
en En
| MEDLINE
| ID: mdl-26714722
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.
Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Ensayo de Inmunoadsorción Enzimática
/
Infecciones por VIH
/
Western Blotting
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VIH-1
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Productos del Gen tat del Virus de la Inmunodeficiencia Humana
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2016
Tipo del documento:
Article
País de afiliación:
Francia