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| | GLO1 Activity Assay |
| Description: | Assays were carried out in 100 mM sodium phosphate, pH 7.0 buffer using 96-well Clear UV Plate (Corning UV Transparent Microplates; catalog #3635). A fresh solution of glutathione (pre-substrate 1, 100 mM) as well as methylglyoxal (pre-substrate 2, 100 mM) was prepared in deionized water. The substrate was prepared by adding 14.5 ml of buffer and 0.99 ml of each pre-substrate components. The substrate mixture was vortexed vigorously for 15 s, then allowed to sit at room temperature for 20 min. Initial well volume was 50 μl containing GLO1 (40 ng) and inhibitor. This protein and inhibitor mixture was incubated for 15–20 min before the addition of substrate. To this was then added substrate (150 μl), yielding a maximum amount of 5% DMSO per well. The enzyme activity was measured using a BioTek Synergy H4 plate reader by measuring absorbance at 240 nmevery 1 min for 8 min. |
| Affinity data for this assay | |
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