DMSO Dimethyl sulfoxide (DMSO) and Dimethylformamide (DMF) are preferred solvents for organic small molecules. They are used to prepare stock solutions with high small molecule concentrations of typically >10 mM, which are often not possible using aqueous buffers. However, DMSO and DMF can negatively affect protein stability and functionality and should thus be kept at a minimal concentration, ideally <5% (v/v).

Although TRIC or Spectral Shift assays are generally not affected by varying DMSO concentrations, it is recommended to keep the DMSO concentration constant throughout the dilution series. There are in general two approaches to set up a Serial Dilution from DMSO-dissolved compounds:

• One can follow the classical approach by preparing the highest concentration of the serial dilution by mixing the compound stock with buffer. This stock is then diluted in buffer with identical DMSO concentration. This approach however bears the risk that the compound might precipitate at the highest concentration(s) (since the added buffer reduces compound solubility), resulting in an erroneous serial dilution and potentially false negative results.
• Alternatively, the compound stock can be serially diluted within 100% DMSO. This ensures that properly dissolved compound is used for the serial dilution, which is completely done in DMSO. The so prepared serial dilution can then be diluted into fluorescent target in assay buffer. In this case, solubility problems of the compound in buffer at highest concentrations do not impact the remaining serial dilution.