Query String: SSA
- ChEMBL_2438369 Binding affinity to NHS-LC-biotin-labeled P2Y14R (unknown origin) immobilized in SSA sensor assessed as dissociation constant by BLI assay
- Biolayer Interferometry (BLI) Binding Assay Biotin labeled BRD proteins (10 μg/ml) in kinetic assay buffer (PBS, pH 7.4, 0.1% BSA and 0.01% Tween-20) were immobilized on Super Streptavidin (SSA) sensors for 15 minutes followed by washing in kinetic buffer for 10 minutes to eliminate any loose non-specific immobilization. In the same 96-well plate serial dilutions of testing compounds with concentrations typically ranging from 0.1-10 times of expected Kd values in the identical assay buffer were prepared. These protein coated sensors were then immersed into the testing compound solutions, starting from the one with the lowest concentration, where compound association occurs and then returned to the fresh buffer for the dissociation. The same operation was repeated for the next solution with higher concentration up to the one with the highest concentration. Identical procedure was performed again with control sensors that were immobilized with SAB4 inactive control protein prepared by following protocols from the manufacturer. Blank buffer controls were included in both BRD protein sensor and inactive protein sensor runs. For each kinetic cycle, kinetic curves for association and dissociation were obtained from raw sensorgrams by using the double reference subtraction protocol included in the analysis program (Data Analysis 7.0) provided by the manufacturer, in which nonspecific interaction and buffer drift were both corrected. The association and dissociation rate constants (kon and koff) were determined using the global fitting protocol in the analysis program based on a reversible 1:1 binding model. The equilibrium association constant (KA) was calculated thereafter. All binding data were collected at 30 degree. Assay plates were kept being shaken at 1000 RPM in the whole experiment time period to avoid mass transport effect.