| Assay Method Information | |
| | Inhibitory Activity of Compounds on the Bromodomain (BRD) of CBP/EP300 |
| Description: | A. Experimental MethodCBP and EP300AlphaScreen Test:1. Prepare 1× concentration of test buffer2. Preparation of compoundThe compound was firstly diluted in a ratio of 1:1000 relative to the final concentration using a Precision automatic sampler (by 3× gradient dilution method):1) 50 μL of compound solution with a storage concentration of 10 mM was transferred to A2 well of a 96-well plate using an Echo automatic sampler.2) 30 μL of DMSO was transferred to well A1, and wells A3 to A12 with Precision™.3) 15 μL solution was transferred from A2 to A3 by Precision, and this operation was sequentially repeated until 15 μL solution was transferred from well A10 to well A11. Thus, a 1:3 gradient dilution was completed.4) The compound plate was centrifuged at a speed of 1000 r/min for one minute.3. Preparation of test plate20 nL of solution was transferred from each concentration well of the compound plate to the test plate using an Echo automatic sampler: from well A1 of the preparation plate to wells A1 and A2 of the test plate, and from well A2 of the preparation plate to wells A3 and A4 of the test plate.4. Binding test of bromodomain (BRD)1) Preparation of 2-fold solution of protein and polypeptideThe protein and polypeptide were dissolved in a 1× concentration of detection buffer.2) 10 μL of protein and polypeptide solution was transferred to each well in columns 3 to 24 in the test plate, and 10 μL of 1× test buffer was transferred to wells in columns 1 to 2 in the test plate as negative control.3) The test plate was centrifuged at a speed of 1000 r/min for one minute.4) The test plate was incubated at room temperature for 15 min.5) A 2-fold receptor and donor solution was prepared by dissolving acceptor and donor beads with one-fold test buffer.6) 2-fold receptor and donor solutions were transferred to the test plate.15 μL of acceptor and donor solution was added and light was avoided.7) The test plate was centrifuged at a speed of 1000 r/min for one minute and then incubated at room temperature for 60 min.5. The Endpoints were Read in EnSpire and Alpha Modes.6. Curve fitting |
| Affinity data for this assay | |
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