| Assay Method Information | |
| | Fluorescence Polarization (FP) Assay |
| Description: | This assay is used to determine whether compounds inhibit the interaction between the BTB domain of BCL6 and a peptide derived from the BCL6 binding domain (BBD) of the SMRT/NCOR2 corepressor proteinCompounds were dissolved in 100% DMSO at 10 mM, assayed fresh, and then stored at −20° C. for repeat studies and future work. The reaction mixture consists of 1.25 uM of the 25 kd BCL6-BTB domain (Thioredoxin-His6-STag-TEV-biotinylation-thrombin-BCL6 amino acids 1-129) plus 20 nM of the peptide probe Ac-GSLVATVKEAGRSIHEIPA (SEQ ID NO:1) with 16 amino-acids (16-mer peptide) from the SMRT BBD (1414-1429) with a Bodipy-TMR fluorescent label on the lysine. The assay buffer was 10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween-20, 3 mM EDTA, and had a final DMSO concentration of 5%. 20 ul of this assay mixture was added to each well of the 384 well plates with the exception of the control wells that contained no protein (for setting the minimum FP value). Compounds were directly sprayed using an HP D300 Digital Dispenser from 10 mM DMSO stocks onto black 384 well plates (greiner bio-one #781900) in a concentration range from 1 uM to 500 uM (10 points in duplicate). The assay was equilibrated for 1 hour prior to reading the FP values (Ex 540 nm/Em 580 nm) with a Perkin Elmer Envision plate reader. The results were curve fitted and IC50 values were calculated using the BioAssay software from CambridgeSoft. Representative compounds displayed IC50<2 uM in this assay (NB: lower limit of this assay was 1 uM). |
| Affinity data for this assay | |
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