| Assay Method Information | |
| | Biochemical KDM5A Inhibition Assay |
| Description: | Using 384-well white Greiner784075 (Greiner), representative compounds were characterized for their inhibition of KDM5A using HTRF technology (Cisbio Bioassays). Briefly, the test compounds, reference compounds, and DMSO control (typical compound concentration range 1 pM-10 μM, final assay concentration (FAC*) of DMSO 0.5%) were added to 384-well plate by the Echo acoustic dispensing platform (Labcyte). Five (5) μl of KDM5A protein (produced by the method described in Nat Chem Biol 12, 531-538 (2016)). (5-20 nM FAC*) in assay buffer (50 mM MES, 50 mM NaCl, 1 mM TCEP, 0.01% v/v Tween 20, 0.03% BSA, pH 6.5) was added to wells and plates were incubated for 10-20 minutes at 25 degrees celsius. Then, 5 μl of alpha-ketoglutaric acid (100 μM FAC*), biotin-labelled H3K4-Me3 substrate (Anaspec Cat #AS-64357-1; 200 nM FAC*), Fe(II)SO4 (100 μM FAC*) and ascorbic acid (2 mM FAC*) in assay buffer was added to wells and plates were incubated for 20 minutes at room temperature. The reaction was stopped with the addition of 10 μl of anti-H3K4-Me2-Eu(K) (Cisbio Bioassays Cat #61KA2KAH; 0.75 nM FAC**)+Streptavidin XL665 (Cisbio Bioassays Cat #610SAXLB; 25 nM FAC**) in HTRF detection buffer (Cisbio Bioassays Cat #62SDBRDF). The mixture was incubated for 30 minutes at room temperature before 340 nm excitation and measurement of dual emission at 620 nm and 665 nm. |
| Affinity data for this assay | |
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