1. Labeling of protein thiols with modified glutathiones
Modified glutathiones were synthesized to study protein S-glutathionylation
Labeling of GAPDH thiols with F-GSH
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.