| Assay Method Information | |
| | Demethylase AlphaScreen Assay |
| Description: | The demethylase AlphaScreen assay was performed in 384-well plate format using white proxiplates (PerkinElmer), and transfer of compound (100 nl) was performed using an ECHO 550 acoustic dispenser (Labcyte). After establishment of suitable purification conditions (see above), enzyme samples showed normal distribution of their activities. All subsequent steps were carried out in assay buffer (50 mM HEPES, pH 7.5, 0.1% (wt/vol) BSA and 0.01% (vol/vol) Tween-20). In brief, 5 μl of assay buffer containing demethylase enzyme at 2× final assay concentration (see Supplementary Table 3c for assay specifics) was preincubated for 15 min with dilutions of compound. The enzyme reaction was initiated by the addition of substrate (5 μl) consisting of L-ascorbic acid (200 μM), 2-OG at 2× final assay concentration (KDM5A-D (10 μM), KDM4C (20 μM), KDM3A (10 μM), KDM6B (20 μM), KDM2A (20 μM)), FAS (2× final assay concentration) and histone H3 substrate peptide (2 |
| Affinity data for this assay | |
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