Extraction of Superior-Quality Plasmid DNA by a Combination of Modified Alkaline Lysis and Silica Matrix
✍ Scribed by Ramakrishna Lakshmi; Vijaya Baskar; Udaykumar Ranga
- Publisher
- Elsevier Science
- Year
- 1999
- Tongue
- English
- Weight
- 161 KB
- Volume
- 272
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
termination were smaller than the symbol designating the mean value of the determination.
Results and Discussion
We found that with our assay conditions, TCEP completely reduced the GSSG that was present within the samples to GSH. Furthermore, the presence of TCEP did not interfere with the labeling of GSH by mBrB nor with the detection of the glutathione-bimane product. This was true for samples consisting of either purified glutathione or acidified extracts of whole blood. As evidence for this, when samples possessing the same molar equivalents of glutathione in either the GSH or GSSG forms were assayed, the resultant fluorescent glutathione-bimane peaks were the same size (Table 1). Also, when GSSG standards with concentrations ranging between 6.125 and 800 M in glutathione were assayed, a linear relationship was obtained between the concentration of glutathione in the sample and the size of glutathione-bimane peak measured (Fig. 1). This linear response was obtained regardless of the sample type, since the addition of defined amounts of GSSG to the acidified extracts of whole blood led to increases in the glutathione-bimane peak that were of the same area as that obtained when that amount of GSSG was assayed by itself (Table 1). Finally, we observed that the glutathione-bimane peaks could be unambiguously attributed and that they were well separated from other fluorescent entities that were generated (Fig. 2).
To date, we have used our modified assay to monitor changes in glutathione resulting from pharmacologic intervention of the HIV disease with N-acetylcysteine (6). We have also used this assay to investigate the redox changes in T cell subsets that occur in the course of HIV infection (manuscript in preparation). In these studies, T cells from uninfected individuals were sorted using fluorescence-activated cell sorting (FACS) to obtain pure populations of the various T cell subsets. The amount of glutathione present in the various purified populations of cells was then determined with our modified assay. A representative chromatogram from these analyses is shown in Fig. 3. We anticipate that the simplicity and general utility of this assay will lead to its wide use.