| Assay Method Information | |
| | AlphaScreen Assay |
| Description: | Materials:EZH2 (non-complexed), His-GST-tagsEED, FLAG-tag4× HMT Buffer 2Anti-FLAG AlphaLISA Acceptor BeadsAlphaScreen GSH Donor BeadsAssay Protocol (Calculated for 384 Wells or 1 Plate)Step One1. Thaw EED and EZH2 proteins on ice.2. Prepare 1× HMT Buffer 2:1.5 mL of 4× HMT Buffer 2+4.5 mL ddH2O.3. Prepare 1.2× EED-EZH2 solution in 1× HMT Buffer 2: 3.4 mL total2.1 uL of EED (0.41 mg/mL, 8.04 uM, 1:1608 dilution)+14 uL of EZH2 (0.24 mg/mL, 2.11 uM, 1:243 dilution)+3.384 mL of 1× HMT Buffer 24. Prepare 1.2× EZH2 solution in 1× HMT Buffer 2 (positive control): 0.168 mL total0.69 uL of EZH2 (0.24 mg/mL, 2.11 uM, 1:243 dilution)+167 uL of 1× HMT Buffer 25. Add 10 μL of 1.2× EED-EZH2 or 1.2× EZH2 solution to each well.6. Add compounds in DMSO via Echo transfer and 30 nL DMSO for control wells.7. Centrifuge at 15,000 rpm for 10 seconds and allow to incubate at rt for 1 hour.Step Two1. Prepare 6× Anti-FLAG and GSH Donor beads in 1× HMT Buffer 2 in the dark: 1 mL total10 uL of Anti-FLAG (1:100 dilution)+5 uL of GSH Donor (1:200 dilution)+985 uL of 1× HMT Buffer 22. Add 2 μL of bead solution to each well.3. Centrifuge at 15,000 rpm for 10 seconds and allow to incubate at rt for 1 hour in the dark.4. Read plate.Final Concentrations:12 μL volume per well8.7 nM EZH25.0 nM EEDDMSO<0.5%Calculations:1. Average EZH2 only wells (positive control) and subtract from all other wells.2. Average EED-EZH2 only wells (negative control) to indicate 100% signal or 0% inhibition.3. Normalize each well to the average 100% signal from step 2.4. Report either as normalized % inhibition or normalized % signal.Use GraphPad to Calculate IC50. |
| Affinity data for this assay | |
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