Ellman Assay

Ellman Assay Markus Hoenicka mhoen@farmr1.med.uth.tmc.edu UT Houston Medical School Integrative Biology and Pharmacology $Date: 2005/01/26 20:24:06 $ $Revision: 1.1.1.1 $ Objective Determination of total sulfhydryl groups, protein-bound sulfhydryl groups, and free sulfhydryl groups in biological samples using DTNB (Ellman's reagent). Sensitivity 50 &mgr;M to 1000 &mgr;M. Sedlak J, Lindsay RH (1968) Anal. Biochem. 25:192-205. Chemicals 5,5'-Dithio-bis(2-nitrobenzoic acid) DTNB 396.3 Sigma D-8130 Ethylenediaminetetraacetate, tetrasodium salt, dihydrate EDTA 416.21 99 % Sigma ED4SS Glutathione GSH 307.3 99 % reduced Sigma G-4251 Methanol p.A. J.T. Baker 8045 Trichloroacetic acid TCA 72 % unknown unknown Triethanolamine-HCl TEA 185.7 Merck 1.08357 Trizma Base 121.1 Sigma T-1503 Devices Microplate reader Multichannel pipettors Repeaters RIA-tube racks Solutions Stock solutions Tris⋅HCl pH 8.2 24.22 g 30 mM 8.32 g 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. Tris⋅HCl pH 8.9 48.44 g 262 mM 8.32 g 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. TEA 1.86 g 20 mM Add MilliQ to make 500 ml. Store at 4°C. TCA 13.9 ml 10 % Stir carefully into 70 ml MilliQ. Add MilliQ to make 100 ml. Store at room temperature. DTNB 29.7 mg 150 &mgr;M Dissolve in 25 ml . Store at 4°C. Fresh solutions Glutathione stock 31.1 mg 20 mM Add to make 5 ml. Prepare fresh daily and keep on ice until used. Procedures GSH standard Add 950 &mgr;l to 50 &mgr;l of to make a 1 mM GSH stock. Prepare the following concentration series: Conc. (&mgr;M) 0 50 100 250 500 1000 GSH 1 mM (&mgr;l) 0 25 50 125 250 500 (&mgr;l) 500 475 450 375 250 0

Total-SH Add the following components to RIA tubes: 20 &mgr;l Sample/Standard 75 &mgr;l 25 &mgr;l 400 &mgr;l Spin down samples at 3000 x g for 5 min at room temperature. Using a multichannel pipettor, transfer 3 x 90 &mgr;l supernatant into a flat-bottom microplate. Measure extinction at 412 nm. Free SH Use a V-bottom microplate to set up the following reaction: 50 &mgr;l Sample/Standard 2 x 25 &mgr;l 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 &mgr;l 20 &mgr;l 50 &mgr;l Supernatant from the TCA precipitation Measure at 412 nm. Protein-bound SH Calculate the difference of the and the .