In affinity screenings (sometimes also secondary screenings), in contrast to Single-Dose Screening, the major goal is not to identify interactors but rather to determine the affinity for known binders. Therefore, affinity screenings are usually a second step, subsequent to an initial single-dose screening.
Serial Dilutions of the ligands of interest are prepared and mixed with a constant concentration of the Target molecule. In TRIC screenings the most common dilution series are 1:1 dilutions of either 12 or 16 points. However, the number of dilution points can be chosen freely in DI.Control software. Depending on the dilution factor and the number of dilutions prepared, a broad or a narrow concentration range can be covered.
The dose response curves resulting from TRIC measurements are then analyzed using either a Kd Fit Model or a Hill Model to determine the dissociation constant (Kd) or the EC50, respectively. Weak Binders can then be distinguished from strong binders and the best candidates for a tight interaction with the target molecule can be identified. To visualize the determined dissociation constants, DI.Screening Analysis software combines a range of dissociation constants into a data bin and shows a histogram on the summary page. The overview chart, shown optionally on the ligands page, can highlight clustering groups of Ligands that might have common chemical properties or common binding modes.
When planning an affinity screening, please familiarize yourself with the term Dilutionseries ID. It is an important determinant for how data is analysed and can also be an important factor when working with Merge Sets.