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.
The original version of this chapter was revised. The erratum to this chapter is available at: DOI 10.1007/978-1-4939-3046-3_26
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Acknowledgement
This work was funded by the CNRS, the ANRS, and Sidaction.
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Rayne, F., Debaisieux, S., Tu, A., Chopard, C., Tryoen-Toth, P., Beaumelle, B. (2016). Detecting HIV-1 Tat in Cell Culture Supernatants by ELISA or Western Blot. In: Prasad, V., Kalpana, G. (eds) HIV Protocols. Methods in Molecular Biology, vol 1354. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3046-3_22
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DOI: https://doi.org/10.1007/978-1-4939-3046-3_22
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3045-6
Online ISBN: 978-1-4939-3046-3
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