| Assay Method Information | |
| | Assay for mIDH1 R132H/S280F |
| Description: | The enzymatic activity of mIDH1R132H/S280F was assessed by measuring the fluorescence of the NADPH. 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 a mIDH1R132H/S280F and NADPH (Santa Cruz, SC202725C) mixture were added to the assay plate for a final concentration of 2 nM and 10 PM, respectively. 5 μL of NADPH alone were added to negative control wells followed by a 1 hour preincubation at room temperature. 5 μL of alphaketoglutarate (Sigma-Aldrich, K1128) were added to the assay plate for a final concentration of 120 μM. Fluorescence of NADPH (excitation: 350 nm, emission 450 nm) was immediately measured kinetically for approximately 90 minutes using the BioTek Synergy Neo plate reader. |
| Affinity data for this assay | |
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