UT Houston Medical School
Integrative Biology and Pharmacology
Version: $Date: 2005/01/26 20:24:06 $ $Revision: 1.1.1.1 $
Status: DRAFT
Determination of total sulfhydryl groups, protein-bound sulfhydryl groups, and free sulfhydryl groups in biological samples using DTNB (Ellman's reagent). Sensitivity 50 µM to 1000 µM.
Sedlak J, Lindsay RH (1968) Anal. Biochem. 25:192-205.
5,5'-Dithio-bis(2-nitrobenzoic acid) (DTNB); MW 396.3; Sigma D-8130
Ethylenediaminetetraacetate, tetrasodium salt, dihydrate (EDTA); MW 416.21; 99 %; Sigma ED4SS
Glutathione (GSH); MW 307.3; 99 %; reduced; Sigma G-4251
Methanol; p.A.; J.T. Baker 8045
Trichloroacetic acid (TCA); 72 %; unknown unknown
Triethanolamine-HCl (TEA); MW 185.7; Merck 1.08357
Trizma Base; MW 121.1; Sigma T-1503
Microplate reader
Multichannel pipettors
Repeaters
RIA-tube racks
| 24.22 g | Trizma Base | final: 30 mM |
| 8.32 g | EDTA | final: 3 mM |
Dissolve in 800 ml MilliQ. Adjust pH with 3 N HCl to 8.2. Add MilliQ to make 1000 ml. Store at room temperature.
| 48.44 g | Trizma Base | final: 262 mM |
| 8.32 g | EDTA | final: 13 mM |
Dissolve in 800 ml MilliQ. Adjust pH with 3 N HCl to 8.9. Add MilliQ to make 1000 ml. Store at room temperature.
| 1.86 g | TEA | 20 mM |
Add MilliQ to make 500 ml. Store at 4°C.
| 13.9 ml | TCA | 10 % |
Stir carefully into 70 ml MilliQ. Add MilliQ to make 100 ml. Store at room temperature.
| 29.7 mg | DTNB | final: 150 µM |
Dissolve in 25 ml Methanol. Store at 4°C.
| 31.1 mg | GSH | 20 mM |
Add TEA to make 5 ml. Prepare fresh daily and keep on ice until used.
Add 950 µl TEA to 50 µl of Glutathione stock to make a 1 mM GSH stock. Prepare the following concentration series:
GSH standard concentration series
| Conc. (µM) | 0 | 50 | 100 | 250 | 500 | 1000 |
|---|---|---|---|---|---|---|
| GSH 1 mM (µl) | 0 | 25 | 50 | 125 | 250 | 500 |
| TEA (µl) | 500 | 475 | 450 | 375 | 250 | 0 |
Add the following components to RIA tubes:
| 20 µl | Sample/Standard |
| 75 µl | Tris·HCl pH 8.2 |
| 25 µl | DTNB |
| 400 µl | Methanol |
Spin down samples at 3000 x g for 5 min at room temperature. Using a multichannel pipettor, transfer 3 x 90 µl supernatant into a flat-bottom microplate. Measure extinction at 412 nm.
Use a V-bottom microplate to set up the following reaction:
| 50 µl | Sample/Standard |
| 2 x 25 µl | TCA |
Spin down for 15 min at 1500 rpm and room temperature and keep the supernatant for later use.
Set up the following reaction in a flat-bottom microplate:
| 200 µl | Tris·HCl pH 8.9 |
| 20 µl | DTNB |
| 50 µl | Supernatant from the TCA precipitation |
Measure at 412 nm.
Calculate the difference of the Total-SH and the Free SH.
Markus Hoenicka: ellman.sgml ($Date: 2005/01/26 20:24:06 $ $Revision: 1.1.1.1 $)