| Assay Method Information | |
| | Assay for WT IDH2 |
| Description: | The enzymatic activity of WT IDH2 was assessed by measuring the fluorescence of the NADPH generated in the enzymatic reaction. The assay was performed in 384 well, white ProxiPlates (PerkinElmer, 6008280) in assay buffer containing 50 mM Hepes (Life Technologies, 15630080), 50 mM KCl (Teknova, P0327), 10 mM MgCl2 (Sigma-Aldrich, M1028), 1 mM DTT (Sigma-Aldrich, 43815), 0.02% BSA (Sigma-Aldrich, A3059) at pH 7.5. The final reaction volume was 10 μL. Stock solutions of the test compounds were prepared in 100% DMSO (Sigma, D2650) and serially diluted 1:3 using 100% DMSO. 50 nL of compound were then transferred to the assay plate using the Beckman Coulter ECHO 650 Acoustic Liquid Handler for a final DMSO concentration of 0.5%. 5 μL of WT IDH2 were added to the assay plate for a final concentration of 0.5 nM. 5 μL of assay buffer alone were added to negative control wells. 5 μL of an isocitrate (Sigma-Aldrich, 58790) and NADP (Sigma-Aldrich, N5755) mixture were added to the assay plate for a final concentration of 5 μM and 15 μM, respectively. Fluorescence of NADPH (excitation: 350 nm, emission 450 nm) was immediately measured kinetically for approximately 15 minutes using the BioTek Synergy Neo plate reader. |
| Affinity data for this assay | |
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