| Assay Method Information | |
| | In Vitro Enzymatic Activity Assay |
| Description: | The activity of recombinant PHGDH was measured by monitoring the reduced nicotinamideadenine dinucleotide (NADH) to nicotinamideadenine dinucleotide (NAD+) change in fluorescence emission at 456 nm. PHGDH (final concentration of 30 ng/ L) was first pre-incubated with enzyme samples in the assay buffer (25 mM HEPES, pH 7.1, 400 mM KCl, 5 M phosphopyridoxa (PLP), 0.5 mM ±-ketoglutarate, 150 M NADH, PSAT1) for 10 min in 96-well plate, then 10 L of DMSO (control) or a small molecule of DMSO solution was added, shaken at 550 rpm for 5 minutes at 25 ° C. and balanced for 5 minutes. In the in vivo testing system of enzyme, each compound was dissolved in DMSO at a final concentration of 5% (v/v), which did not affect the assay signal. The reaction was started by adding L-phospho-O-serine (Pser) solution. The UV-visible microplate reader was used to monitor the change of NADH consumption at 456 nm with time. Protein activity was assessed by using an initial rate of reaction within 30 s, at which time NADH consumption was linear over time. |
| Affinity data for this assay | |
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