In affinity screenings (sometimes also secondary screenings), in contrast to Single-Dose Screening, the primary goal is not to identify interactors but to determine the affinity for known binders. Therefore, affinity screenings are usually a second step after an initial single-dose screening.

Serial Dilutions of the ligands of interest are prepared and mixed with a constant Target molecule concentration. The most common dilution series used in screenings is 1:1 of 12 or 16 points. However, the number of dilution points can be freely chosen in DI.Control. Depending on the dilution factor and the number of dilutions prepared, one can cover either a broad or a narrow concentration range.

The dose-response curves resulting from Dianthus 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 be distinguished from strong binders so that you can rank the best candidates with a tight interaction. To visualize the determined dissociation constants, DI.Screening Analysis combines a range of dissociation constants into a data bin and shows a histogram on the summary page. The overview chart, optionally shown on the ligands page, can potentially highlight clustering groups of Ligands that might have common chemical properties or binding modes.

When planning an affinity screening, please familiarize yourself with the term “DilutionSeries ID.” It is an essential determinant for data analysis and an important factor when working with Merge Sets.

How to program DI.CO to read an affinity screening:

After plating your ligands and incubating them with the labeled target in the optimal buffer, one can use the “Quick Start” option or the “Import” option in the “Affinity Screening” sub-menu options.

Using the “Quick Start” link:

Click on the “Quick Start” option from the Affinity Screening menu, then select the wells to measure, followed by the “Add Selected Wells” on the menu on top of the plate draw. Alternatively, the “Add All wells” option will select a full plate. After this, select the Laser On-time. The default measurement Is Spectral Shift only without TRIC. If one wants to add TRIC to the measurement, click the 3s or 5s option to read Spectral Shift and TRIC. Finally, select the Excitation power and a Delayed Experiments option (if desired, to pre-incubate in the instrument before measurement). Usually, to compare results, choosing an excitation power during the assay setup and optimization is best, and then leaving that value constant for all the following measurements with the same target.

One can then annotate the table during or after measuring the plate if needed.