We believe this site might serve you best::

United States

United States

Choose language: English

Promega's Cookie Policy

Our website uses functional cookies that do not collect any personal information or track your browsing activity. When you select your country, you agree that we can place these functional cookies on your device.

Homogeneous Add-Mix-Read Assay for Total and Oxidized Glutathione Poster

SEARCH POSTERS

Abstract

John Shultz, Mary Sobol, Nancy Murphy, Jolanta Vidugiris
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711

A wide variety of compounds can induce intracellular formation of reactive oxygen species (ROS) and the resulting cellular damage can eventually lead to apoptosis or necrosis. Measuring the ratio of reduced to oxidized glutathione (GSH/GSSG ratio) is a way to determine how such compounds alter the redox potential in the cell. A new method is presented for rapid determination of the GSH/GSSG ratio in mammalian cells in multiwell tissue culture plates. The method is based on the formation of luciferin from a glutathione S-transferase catalyzed reaction using GSH and a luciferin precursor. The method utilizes a cell lysate generated directly in the wells of the cell culture plate and does not require protein removal steps. In the reaction to measure total glutathione, DTT is added to convert all the glutathione in the reaction to the reduced form. In the reaction to measure oxidized glutathione (GSSG), free sulfhydral groups are inactivated with NEM leaving the GSSG intact. The inactivation step is followed by a reducing step that converts the GSSG to GSH for quantification in the luminescent reaction. Since the reaction of free sulfhydral groups takes place immediately upon cell lysis, additional formation of GSSG from air oxidation of GSH is prevented. The assay takes less than two hours from the time a plate of cells is ready for measurement and can be performed using less than 1000 cells per well. The performance of the method is demonstrated by following the effects of compounds such as menadione, ethacrynic acid and chlorodinitrobenzene on the levels of GSH and GSSG in cells. This is a fast, easy, and sensitive assay for identification of compounds that alter the GSH/GSSG ratio.

Document Icon Download the poster PDF (620kb)

  • Part# PS121
  • Printed in USA.
Choose your country

Americas

Brazil
Brazil
Canada
Canada
United States
United States

Pacific Asia

Australia
Australia
India
India
Japan
Japan
Korea, Republic of
Korea, Republic of
Singapore
Singapore

Europe

Austria
Austria
Belgium
Belgium
Denmark
Denmark
Estonia
Estonia
Finland
Finland
France
France
Germany
Germany
Iceland
Iceland
Italy
Italy
Luxembourg
Luxembourg
Netherlands
Netherlands
Norway
Norway
Poland
Poland
Spain
Spain
Sweden
Sweden
Switzerland
Switzerland
United Kingdom
United Kingdom