| Assay Method Information | |
| | Determination of the value of binding affinity constant (KO between the compound and BRD4 BD1 protein |
| Description: | The purity of BRD4 BD1 protein used in the experiment was greater than 95%, and the protein concentration was 43.4 uM. The 96-well plate was purchased from Corning (black, #3694). The multifunctional microplate reader was a product of TECAN, model: SPARK 10M. Buffer: 100 mM potassium phosphate (pH 6.5), 2% ethylene glycol (Sigma) and 0.01% Trition X-100 (Sigma). The experimental water was Millipore-Q pure water.The specific experimental steps were as follows.First, the compound to be tested was dissolved in ethylene glycol to prepare into a 10 mM standard stock solution. Subsequently, the standard stock solution of the compound to be tested was diluted into a working sample solution with the buffer in an EP tube and ready for use. The concentration of the prepared working sample solution was 5 times of the highest sample concentration required on the test plate (5×test compound solution).40 λL of a 5× test compound solution of a sample A was added to wells B1-B3 of a 96-well plate, and 40 μL of a 5× test compound solution of a sample B was added to wells B7-B9 of the 96-well plate, respectively. 20 uL of the buffer was added to the remaining wells, except for wells B1-B3 and B7-B9. Then, 20 uL of a solution was taken from wells B1-B3 to C1-C3, and this 2-fold dilution was repeated from C1-C3 until H4-H6; in the same way, 20 uL of a solution was taken from B7-B9 to C7-C9, this 2-fold dilution was repeated from C7-C9 until H10-H12. Finally, 80 uL of a mixed solution containing 2.5 nM Tracer and 37.5 nM BRD4 BD1 protein was added to each well. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |