Search and Browse
Download
Enter Data
|
Assay Method Information |
|
| | Scintillation Proximity Assay (SPA) |
| Description: | Compounds contained herein were evaluated for their ability to inhibit the methyltransferase activity of EZH2 within the PRC2 complex. Human PRC2 complex was prepared by co-expressing each of the 5 member proteins (FLAG-EZH2, EED, SUZ12, RbAp48, AEBP2) in Sf9 cells followed by co-purification. Enzyme activity was measured in a scintillation proximity assay (SPA) where a tritiated methyl group is transferred from 3H-SAM to a lysine residue on Histone H3 of a mononucleosome, purified from HeLa cells. Mononucleosomes were captured on SPA beads and the resulting signal is read on a ViewLux plate reader. Compound Preparation 1. Prepare 10 mM stock of compounds from solid in 100% DMSO. 2. Set up an 11-point serial dilution (1:3 dilution, top concentration 10 mM) in 100% DMSO for each test compound in a 384 well plate leaving columns 6 and 18 for DMSO controls. 3. Dispense 100 nL of compound from the dilution plate into reaction plates (Grenier Bio-One, 384-well, Cat#784075). Part B. Reagent Preparation Prepare the following solutions: 1. 50 mM Tris-HCl, pH 8: Per 1 L of base buffer, combine 1 M Tris-HCl, pH 8 (50 mL) and distilled water (950 mL). 2. 1× Assay Buffer: Per 10 mL of 1× Assay Buffer, combine 50 mM Tris-HCl, pH 8 (9958 uL), 1 M MgCl2 (20 uL), 2 M DTT (20 uL), and 10% Tween-20 (2 uL) to provide a final concentration of 50 mM Tris-HCl, pH 8, 2 mM MgCl2, 4 mM DTT, 0.002% Tween-20. 3. 2× Enzyme Solution: Per 10 mL of 2× Enzyme Solution, combine 1× Assay Buffer and PRC2 complex to provide a final enzyme concentration of 10 nM. 4. SPA Bead Suspension: Per 1 mL of SPA Bead Suspension, combine PS-PEI coated LEADSeeker beads (40 mg) and H2O (1 mL) to provide a final concentration of 40 mg/mL. 5. 2× Substrate Solution: Per 10 mL of 2× Substrate Solution, combine 1× Assay Buffer (9728.55 uL), 800 ug/mL mononucleosomes (125 uL), 1 mM cold SAM (4 uL), and 7.02 uM 3H-SAM (142.45 uL; 0.55 mCi/mL) to provide a final concentration of 5 ug/mL nucleosomes, 0.2 uM cold SAM, and 0.05 uM 3H-SAM. 6. 2.67× Quench/Bead Mixture: Per 10 mL of 2.67× Quench/Bead Mixture, combine ddH2O (9358 uL), 10 mM cold SAM (267 uL), 40 mg/mL Bead Suspension (375 uL) to provide a final concentration of 100 uM cold SAM and 0.5 mg/mL SPA beads. Part C. Assay Reaction in 384-well Grenier Bio-One Plates Compound Addition 1. Dispense 100 nL/well of 100× Compound to test wells (as noted above). 2. Dispense 100 nL/well of 100% DMSO to columns 6 & 18 for high and low controls, respectively. Assay 1. Dispense 5 uL/well of 1× Assay Buffer to column 18 (low control reactions). 2. Dispense 5 uL/well of 2× Enzyme Solution to columns 1-17, 19-24. 3. Spin assay plates for 1 min at 500 rpm. 4. Stack the assay plates, covering the top plate. 5. Incubate the compound/DMSO with the enzyme for 30 min at room temperature. 6. Dispense 5 uL/well of 2× Substrate Solution to columns 1-24. 7. Spin assay plates for 1 min at 500 rpm. 8. Stack the assay plates, covering the top plate. 9. Incubate the assay plates at room temperature for 1 hour. Quench/Bead Addition 1. Dispense 5 uL/well of the 3× Quench/Bead Mixture to columns 1-24. 2. Seal the top of each assay plate with adhesive TopSeal. 3. Spin assay plates for 1 min at 500 rpm. 4. Equilibrate the plates for >20 min. Read plates 1. Read the assay plates on the Viewlux Plate Reader utilizing the 613 nm emission filter with a 300 s read time. Reagent addition can be done manually or with automated liquid handler. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |
|
Home |
| |
Search |
| |
Deposit |
| |
SiteMap |
| |
About us |
| |
Email us |
| |
Info |
|
©2000 BindingDB. All rights reserved. |
|||||||||||||