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In order to measure oxidized glutathione (GSSG), you must eliminate GSH in the cell extract by treating with 4-VP, a compound which alkylates SH groups. It is important to run a 4-VP blank, and we recommend that designated samples be set aside for GSSG measurements. For the standards, 4-VP should be added to the concentrated 4 µM GSSG, incubated for 1 hour at room temperature, then serially diluted according to the protocol. For test samples, first dilute your samples to the appropriate concentration, then add the 4-VP (1 µl 2M 4-VP per 50 µl of sample) and incubate for 1 hour at room temperature. Essentially, the 4-VP treatment is part of your sample preparation. Once you have completed incubation, proceed with the serial dilution of your standards (containing 4-VP), add your samples to the plate, then the reaction mix and take absorbance readings as soon as possible.