A stepwise dilution of Ligand in Buffer. Typically, a serial 1:1 dilution (dilution factor 2 in DI.Control) is performed by transferring one volume of ligand solution to an equal volume of buffer, mixing, and repeating this step. This way, the ligand concentration is reduced by 50% in each dilution step. The dilution factor can be freely determined by the user and is often adapted to obtain more datapoints for a transition phase or to cover a broader concentration range with fewer dilution points. For ligands that are stored in DMSO, dilution is either performed in a way that maintains a constant level of DMSO throughout the dilution series or they are diluted in 100% DMSO and then each dilution point is diluted into buffer individually.

General considerations

• Never prepare less than 20 μl of sample.
• Never prepare small volumes (e.g. 20 μl) in large micro reaction tubes (e.g. 500 μl or more) and then transfer into the Dianthus microwell plate. The high surface to volume ratio leads to surface adsorption even for well-behaved proteins. Always use the smallest micro reaction tubes possible (e.g. PCR tubes) or other microwell plates and excess of volume to transfer to the Dianthus plate (e.g., 30 μl).
• Always mix small volumes with a pipette. Do not vortex protein samples.
• Avoid any buffer dilution effects. The buffer in tube 1 and the buffer in the tubes 2-16 must be the same. Otherwise, you one could bias the measured data with a gradient in salt, DMSO, glycerol or other additives. Although TRIC and Spectral Shift are very robust against such dilution effects, it could affect the measurements.
• All wells that are part of a serial dilution should have the same Dilutionseries ID. This is important for all Affinity Screening experiment templates.