Primary screenings are usually conducted in a single-dose setup and are best and most efficiently analyzed using DI.Screening Analysis software. Each Ligand is tested at a single, high concentration. This experimental setup provides a simple yes/no answer for ligand binding, and is suited for screening a larger number of ligands compared to the quantitative Affinity Screening. If the ligand binds the Target, it will change the target TRIC signal and is therefore a Potential Hit . The used high ligand concentration can be adjusted in order to pre-select for ligands with desired affinities, e.g. limiting this concentration to 50 μM omits the detection of weak binders with significantly lower affinities.
A single-dose screening requires the following controls and setup details:
- A reference sample. The reference sample includes the target in the final assay buffer, but no ligand. The reference is required to set thresholds for hits and Non-Binders and to control for signal stability over the duration of the screening. A reference sample must be measured regularly (several times per plate) for best results. The reference sample is often equivalent to a DMSO control.
- A positive control. The positive control interaction includes target and a ligand that is a known binder. This is to control for target functionality (its capability to bind ligands) over the duration of the screening.
- If possible, ligands should be run in duplicate.
When planning a single- dose screening, familiarize yourself with the term Normalization ID . It is an important determinant for how data is analysed in DI.Screening Analysis software.