NWLSS™ NWK-GSH01 High Sensitivity Protocol
Revision 08/29/06

The standard method of the NWLSS™ NWK-GSH01 Assay is designed to conveniently measure total gultathione (GSH + GSSG) in most common samples such as whole blood or liver where the GSH concentration is in the 1-10 mM range. A 20-fold greater sensitivity can easily be achieved by modifiying the standard method of the NWLSS GSH assay.

As can be seen in the figures below, the reaction rate curves and calibration curve obtained using the high sensitivity modification maintain the required linearity.

GSH01 Modified Method for High Sensitivity Prepare samples as appropriate Dilute the GSHeq calibrator 1/400 and 1/800 (1 µM and 0.5 µM) in Assay Buffer Add 50 µL calibrator or sample to microplate well Add 50 µL DTNB Add 50 µL GR Incubate for 2-3 minutes Add 50 µL NADPH Monitor the 405 nm absorbance • Interval = 2-3 minutes • Duration = 30 miuntes

Calibration Parameters • Slope 0.0207 • Intercept 0.0028 • r2 0.9993 • Syx 0.0003 • MLD* in Reaction 0.0253 *Method Limit of Detection = 2Syx/Slope

Why does this work?

The GSH recycling method is limited by the available concentrations of NADPH and the absorbance range of the plate reader. As the reaction proceeds, the consumption of NADPH is proportional to the GSH concentration and as the NADPH becomes limiting, the reaction rate slows and the curve loses linearity. Reducing the concentration of the calibrators allows the reaction duration to be extended to acheive sufficient ΔA₄₀₅ to confidently determine the rates.