| Assay Method Information | |
| | Competitive HTRF Assay |
| Description: | The general procedure of the competitive HTRF assay for the affinity to CBP recombinant proteins is as follows: 0.05% Tween-20 and 1 mM of TCEP were added immediately before the assay, and the assay was performed in a buffer consisting of 0.1 mg/ml BSA, 50 mM of HEPES and 5 mM of NaCl of pH 7.5. 2.5 μL of compound solution in a determination buffer with 4% DMSO and 5 μL of CBP solution in a determination buffer were added to a white low-volume 384-well microtiter plate, incubated at room temperature for 20 min, and then incubated at room temperature for 40 min by adding 2.5 μL of biotin-labeled ligand solution to the determination buffer. The final concentrations of CBP, biotin-labeled ligand and DMSO were 5 nM, 50 nM and 1%, respectively. Then, 5 μL of MAb Anti 6HIS—Eu cryptate Gold and 5 pt of Streptavidin-XL665 in detection buffers from the manufacturer were added to the mixture, and the mixture was incubated for another 60 min. The final concentration of Streptavidin-XL665 was 12.5 nM, and MAb Anti 6HIS—Eu cryptate Gold was diluted according to the final concentration provided by the supplier. The multimode reader Spark from TECAN (Mannedorf, Switzerland) was used to read the plate to detect the homogeneous time-resolved fluorescence intensity of two groups, in which the excitation wavelength was 320 nm and the emission wavelengths were 665 nm and 620 nm. The IC50 value of the inhibitor was obtained by fitting fluorescence intensity ratios at 665 nm/620 nm with respect to the inhibitor concentration in an S-shaped dose-response curve by using Prism 7 (La Jolla, 15 CA). |
| Affinity data for this assay | |
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