Figure 8.

Infuence of the storage conditions on Tat functional stability. Freshly reconstituted Tat (in 20 mM Tris, pH 8, and 1 mM DTT) was divided into several aliquots and stored at different temperatures (RT, 4°C, -20°C, and -70°C) and incubated for different periods (0, 8, 15, 30 and 60 days). As a control, Tat was incubated at these different temperatures in a lyophilized form. (A) SDS-PAGE analysis. A portion of the aliquots was mixed with SDS-PAGE buffer, boiled and the samples were resolved on a 15% SDS-PAGE gel. M, protein molecular weight standards (# SM0431, MBI Fermentas); RT, room temperature. In parallel, portions of the same aliquots were used to evulate Tat's potential (B) to stimulate TNF-α production from primay monocytes (C) to rescue a defective provirus from HLM-1 cells and (D) to measure free sulfhydryl groups in Tat using DTNB assay. The experiment was performed twice and the data presented are from one representative experiment. Data are presented as mean value of triplicate wells ± 1 S.D.