| Assay Method Information | |
| | Primary Assay Used to Determine Potency of MEN1 Activity Inhibition |
| Description: | Compound activity was determined using recombinant MEN1 protein (Creativebiomart, Cat #MEN1-35H) and a custom fluorescein-labeled MLL4-43 peptide (Eton Bioscience Inc.). Interaction between MEN1 and MLL4-43 in the presence of compounds was determined by fluorescence polarization assay using a Microplate Reader ClarioStar Plus. The reaction was carried out in assay buffer (50 mM TRIS-HCl pH 7.4-7.6, 50 mM NaCl, 1 mM DTT, 0.1 mg/ml BSA). The compounds were dispensed on a 384 well Diamond Well Plate (Axigen, Cat #P-384-120SQ-C-S) using the Biomek FX liquid handling system at 100× solutions of compounds in DMSO. 2×MEN1 mix (final concentration of MEN1 10 nM) was prepared in Assay buffer and 10 μl of mixture per well was added into 384 w white Reaction plate with NBS (Corning, Cat #4513). 10 μl of Assay buffer w/o MEN1 was used for negative control. Plates were centrifuged for 1 min at 100 g. Next step the Compounds were added to Reaction plate using Biomek station via following steps: 3 μl of 100× compounds (in DMSO) were mixed thoroughly with 27 μl Assay Buffer, then 2 μl of this mixture was added to Reaction plate with 10 μl of MEN1 mix. Plates were centrifuged for 1 min at 100 g and incubated for 20 min at room temperature. Next 8 μL of MLL4-43 peptide per well was added to final concentration of MLL 0.5 nM. Plates were incubated for 1 hour at room temperature. Then fluorescence polarization was measured using Microplate Reader. |
| Affinity data for this assay | |
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