2012 Promega catalogue

Life
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2012
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Cell Signaling
Glutathione Measurement
44
GSH/GSSG-Glo Assay
Product Size Cat.# Price ($)
GSH/GSSG-Glo Assay 10 ml V6611 755.00
For Research Use Only. Not for Use in Diagnostic Procedures.
50 ml V6612 3022.00
Description: The GSH/GSSG-Glo Assay is a luminescence-based system
for the detection and quantification of total glutathione (GSH +GSSG), GSSG
and GSH/GSSG ratios in cultured cells. A change in GSH levels is important
in the assessment of toxicological responses and is an indicator of oxidative
stress, potentially leading to apoptosis or cell death. The assay provides a
simple multiwell-plate format where stable luminescent signals are correlated
with either the total GSH or the GSSG concentration of a sample directly in
culture wells. Both total glutathione and GSSG determinations are based on
the reaction where GSH-dependent conversion of a GSH probe, Luciferin-NT, to
luciferin by a glutathione-S-transferase enzyme is coupled to a firefly luciferase
reaction. Light from luciferase is dependent on the amount of luciferin formed,
which is in turn dependent on the amount of GSH present. This makes the
luminescent signal proportional to the amount of GSH. Determination of total
glutathione and GSSG are performed in parallel reactions. In one configuration
the assay reagents measure total glutathione using a reducing agent that
converts all the glutathione, GSH and GSSG in a cell lysate to the reduced form,
GSH. In a second configuration the assay reagents are used to measure only
the oxidized form, GSSG. In this case, a reagent is added that blocks all the
GSH while leaving the GSSG intact. This blocking step is followed by a reducing
step that converts the GSSG to GSH for quantification in the luminescent reaction.
Because the assays are performed directly on cells in culture wells, loss of
GSH or GSSG is minimized, reducing variability.
Features:
• Physiologically Relevant GSH/GSSG Ratios: Actual levels of total glutathione
and GSSG are measured directly in cell-culture wells, minimizing
the loss of GSH and GSSG, compared to conventional assays that require
upfront sample preparation and indirect GSSG calculation.
• More Robust Performance: Bioluminescent technology and a simple
protocol minimize sample handling, reducing variability.
• Simplified Protocol: Assay reagents are added directly to cells cultured
in multiwell plates. The homogeneous add-mix-read format eliminates
time-consuming sample deproteination and centrifugation steps required
of conventional assays.
• Greater Sensitivity: Fewer cells are required in these assays than in
conventional assays because of the enhanced sensitivity.
• Faster Results: The homogeneous add-mix-read protocol minimizes
hands-on time, and the bioluminescence technology minimizes incubation
time.
• Adaptable to Automation: The glow-type signal is stable, with a half-life
greater than two hours, and the protocol is adaptable to automation in 96-
and 384-well plates.
• No Fluorescence Interference: Using luminescence readout eliminates
the fluorescent interference between reagents and test compounds sometimes
seen in fluorescence assays. Such overlap can confound analysis
and present misleading or irrelevant data.
Storage Conditions: Store at –20C protected from light.
Protocol Part#
GSH/GSSG-Glo Assay Technical Manual TM344
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