| Assay Method Information | |
| | Enolase Enzymatic Assays |
| Description: | Enolase activity was measured by two different methods: a fluorometric NADH-linked assay or adirect spectrophotometric assay via formation of PEP. In the fluorescent assay,enolase activity was measured via NADH oxidation in a pyruvate kinase-dehydrogenase coupled assay. The assay is conducted in 10 mM KCl, 5 mM MgSO4, and 100 mM triethanolamine at pH 7.4, with 400 uM NADH and 2 mM ADP. 2-PGA, pyruvate kinase (PK) and lactate dehydrogenase(LDH) are provided in excess, with conversion of 2-PGA to PEP by enolase being rate limiting. PEP (with ADP) is substrate of PK; pyruvate formed by thisreaction is linked to NADH oxidation by LDH. Enolase activity is determinedby fluorescence measurement of oxidation of NADH by excitation at 340 nmand emission at 460 nm. The substrate concentration was 5 mM 2-PGA unlessotherwise indicated. Fluorescence was measured using an Omega FluorescencePlate Reader (BMG Labtech). Alternatively, enolase activity was measured directly by the appearance of PEP from 2-PGA via absorption at 240 nm. The assay medium was the same, except that all the auxiliary reagents (PK or LDH, NADH and ADP) are omitted. Both assays were conducted in a 96-well plate format, with the direct assay performed in UV-transmissible plates. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |