| Assay Method Information | |
| | Enzyme Assay of Inhibition Against MLL1-WDR5 Protein-Protein Interactions |
| Description: | WDR5 TR-FRET Assay Procedure: Stock compounds were transferred to the assay plate by Echo Liquid Handler. Reactions were performed in the assay buffer (1×PBS, 300 mM NaCl, 0.5 mM TCEP, 0.1% CHAPS) containing 5 nM WDR5 protein, 10 nM peptide (Ac-ARTEVHLRKS-[Ahx-Ahx] [C]-Alexa Fluor 488-NH2) and 0.25 nM Tb-anti His antibody (Tb-Ab) in 384-well white plate (PerkinElmer) with a final volume of 20 μl. Compounds were incubated with WDR5 protein for 30 min at room temperature. Plates were covered, protected from light and incubated for 60 min at room temperature after adding the peptide and Tb-Ab. EnVision Multimode Plate Reader (PerkinElmer) was used for the TR-FRET assay with excitation wavelength at 340 nm and emission wavelength at 495 and 520 nm. The ratio of the 520/495 wavelengths were used to assess the degree of the FRET signal. IC50 was calculated by fitting the inhibition data using XLfit software to sigmoidal dose-response model. |
| Affinity data for this assay | |
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