| Assay Method Information | |
| | Fluorescamine Assay |
| Description: | In a standard optimized reaction, SpPgdA was added to a microcentrifuge tube and incubated at 30 °C for 5 min. Chitotriose was added as a substrate to obtain the desired final substrate concentration. Total reaction volume was 50 µL and contained SpPgdA (2 µM) and cobalt(II) chloride (5 µM) in HEPES (25 mM, pH 7.0). Immediately after addition of substrate, the sample was incubated at 30 °C. Aliquots (10 µL) were removed from the reaction at 75 or 100 s intervals for up to 5 min. Each aliquot was immediately added to quenching buffer (20 µL, 0.4 M borate, 100 mM EDTA, pH 9.0), followed by the addition of fluorescamine in DMF (10 µL, 2 mg/mL). This labeling reaction was allowed to occur for 10 min at room temperature before the addition of DMF/H2O, and the fluorescence was measured (excitation = 360 nm, emission = 460 nm). |
| Affinity data for this assay | |
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