Extraction of superior-quality plasmid DNA by a combination of modified alkaline lysis and silica matrix.

We found that with our assay conditions, TCEP comletely reduced the GSSG that was present within the amples to GSH. Furthermore, the presence of TCEP id not interfere with the labeling of GSH by mBrB nor ith the detection of the glutathione–bimane product. his was true for samples consisting of either purified lutathione or acidified extracts of whole blood. As vidence for this, when samples possessing the same olar equivalents of glutathione in either the GSH or SSG forms were assayed, the resultant fluorescent lutathione–bimane peaks were the same size (Table ). Also, when GSSG standards with concentrations anging between 6.125 and 800 mM in glutathione were ssayed, a linear relationship was obtained between he concentration of glutathione in the sample and the ize of glutathione–bimane peak measured (Fig. 1). his linear response was obtained regardless of the ample type, since the addition of defined amounts of SSG to the acidified extracts of whole blood led to ncreases in the glutathione–bimane peak that were of he same area as that obtained when that amount of SSG was assayed by itself (Table 1). Finally, we oberved that the glutathione–bimane peaks could be nambiguously attributed and that they were well seprated from other fluorescent entities that were generted (Fig. 2). IG. 3. Representative chromatogram obtained from the analysis f the glutathione present within FACS sorted cells. Approximately 3 10 purified naı̈ve CD4 human T cells (CD4, CD45, CD62L) ere FACS sorted, washed free of serum, and resuspended in 100 ml f 5% sulfosalicylic acid plus 50 mM DTT. The samples were then nalyzed with our modified mBrB assay. The peak at 6.5 min. is the lutathione–bimane peak. hanges in glutathione resulting from pharmacologic ntervention of the HIV disease with N-acetylcysteine 6). We have also used this assay to investigate the edox changes in T cell subsets that occur in the course f HIV infection (manuscript in preparation). In these tudies, T cells from uninfected individuals were sorted sing fluorescence-activated cell sorting (FACS) to obain pure populations of the various T cell subsets. The mount of glutathione present in the various purified opulations of cells was then determined with our modfied assay. A representative chromatogram from these nalyses is shown in Fig. 3. We anticipate that the implicity and general utility of this assay will lead to ts wide use.