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Thiols and disulfides: Difference between revisions
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=== Method according to Riddles: determination of thiols with DTNB === | === Method according to Riddles: determination of thiols with DTNB === | ||
Dilute protein in 1 ml of 6 M Guanidine-HCl, 50mM Tris-HCl, pH 8.3, 1 mM EDTA to a final concentration of 10-40 µM thiols. Add 10 µl of 10 mM DTNB (Dithionitrobenzoic acid; Ellman’s reagent) in 100 mM Tris-Cl, pH 7.6. | Dilute protein in 1 ml of 6 M Guanidine-HCl, 50mM Tris-HCl, pH 8.3, 1 mM EDTA to a final concentration of 10-40 µM thiols. Add 10 µl of 10 mM DTNB (Dithionitrobenzoic acid; Ellman’s reagent) in 100 mM Tris-Cl, pH 7.6. The DTNB is cleaved by the thiol and a mixed disulfide of one NTB moiety with the Cys thiol is formed.The other NTB moiety has an intense absorption band at 412 nm. Extinction coefficient is 13,600 M-1 cm-1 per thiol. Make blank with buffer and DTNB, since the absorption band of DTNB tails to 412 nm. | ||
If the thiols in the protein are oxidizing very fast keep the protein at low pH, which will keep the thiol protonated. Only the thiolate is oxidizing very fast. At low pH the assay with DTNB does not work. | If the thiols in the protein are oxidizing very fast keep the protein at low pH, which will keep the thiol protonated. Only the thiolate is oxidizing very fast. At low pH the assay with DTNB does not work. | ||