BIOL 239 |
| Thiol status is an important parameter to reflect cell oxidative stress. Ellman's reagent [5,5 '-dithiobis-(2-nitrobenzoic acid), DTNB] is a thiol specific reagent and has been widely used for thiol quantification. Quantification of protein thiols and disulfides by DTNB has been primarily achieved by determination of 5-thio-2-nitrobenzoic acid (TNB), a product generated after DTNB reacts with a thiol, spectrophotometrically at 412 nm. One of the major drawbacks of this assay is the interference by compounds which exhibit absorption at 412 nm. We would like to report an HPLC method for the determination of TNB. In developing the HPLC conditions, TNB was found to be detected much more sensitively at 314 nm than at 412 nm as used in the spectrophotometric assay. The results of this method used for the quantification of thiols and disulfides from cell samples and a comparison with the results obtained from the spectrophotometric assay will be presented. |
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Frontiers in Chemical Biology
5:00 PM-7:00 PM, Wednesday, August 22, 2007 BCEC -- Exhibit Hall - B2, Poster
Division of Biological Chemistry |