Anti-DTNB monoclonal antibody Made to order

The conformation changes in S-100a.a' and S-100b caused by K+-binding were determined by several methods in the apo-state and in the Ca2+-bound state. The reactivities of the cysteine (Cys) residues in S-100a.a' and S-100b to a thiol-specific reagent, 2,2'-dinitro-5,5'-dithiodibenzoic acid (DTNB), were not affected by the K+-binding. On the contrary, the reactivities of those in Ca2+/S-100a.a' and Ca2+/S-100b toward DTNB were enhanced or decreased, respectively, by the K+-binding. The conformation changes in S-100b, Ca2+/S-100a.a' and Ca2+/S-100b caused by the K+-binding were also detected using a fluorescence environmental probe, 2-p-toluidinonaphthalene-6-sulfonate (TNS). A clear difference in the decrease of the TNS fluorescence intensity obtained by the K+-binding between S-100a.a' and S-100b was demonstrated in the Ca2+-bound state. The measurements of the difference absorption spectrum of S-100a.a' obtained by the K-binding showed a positive spectrum, while those of S-100b showed a negative spectrum, regardless of the Ca2+-binding.

Objective: Sulfhydryl groups of protein, glutathione and free cysteine are readily oxidized to disulfides by oxidants, and are important indicators of oxidative stress. Our aim was to apply the Ellman method for assay of sulfhydryl groups to microplate scale and to determine this method's analytical performance. Method: Reaction products produced by methanolic 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) were measured at 412 nm using a multimode microplate reader that contains a monochromator. Results: In standard curve, linearity was obtained in the range of 0.01-4.0 mM concentration of reduced glutathione concentration. Reproducibility of the assay within-run, within-day and between-days were detected as between 6.2%-7.5%. Recovery of glutathione was 91-113%. The method could also be used with tissue homogenates, after solubilization by sodium dodecyl sulfate. Conclusions: The Ellman's reaction can be applied at the microplate scale with good analytical performance and is a quick and reliable method for use with numerous plasma and tissue samples.