Labeling of protein thiols with modified glutathiones

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Labeling of protein thiols with modified glutathiones Modified glutathiones were synthesized to study protein S-glutathionylation Labeling of GAPDH thiols with F-GSH 1 2 3 GAPDH was treated as shown below, subjected to SDS-PAGE under nonreducing conditions and the gel was photographed under UV light. F-GSH: fluorescein-labeled GSH B-GSH: biotinylated GSH F-GSSG-F and B-GSSG-B are the oxidized forms. 1. Thiol-disulfide exchange with F-GSH 2. Reduction of mixed disulfide with DTT 3. Increased yield of mixed disulfide with H2O2 Labeling of protein thiols with B-GSH ADH CK GAPDH papain Proteins were treated with B-GSH and H2O2, separated by SDS-PAGE under nonreducing conditions and transferred to PVDF. Biotinylated proteins were detected using avidin-HPR and a chemiluminescent substrate. Addition of DTT abolished labeling for all proteins tested.