1 / 1

Protein Thiol Labeling with Modified Glutathiones Study

Synthesis and analysis of modified glutathiones for protein S-glutathionylation. Labeling of GAPDH thiols with F-GSH, thiol-disulfide exchange, reduction of disulfides, and increased yield with H2O2 treatment. Detection via SDS-PAGE and transfer to PVDF membrane.

chava
Télécharger la présentation

Protein Thiol Labeling with Modified Glutathiones Study

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 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 Labeling of protein thiols with B-GSH 2. Reduction of mixed disulfide with DTT ADH CK GAPDH papain 3. Increased yield of mixed disulfide with H2O2 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.

More Related