Assay Development and Miniaturization

High-quality assays are essential for generating reliable screening data that can lead to the discovery of high-quality chemical probes or drug candidates.

In a high-throughput setup, assays should provide a large assay window (signal-to-background ratio) that warrants sufficient statistical power to reliably identify true positive hits. The typical signal-to-background ratio should be greater than 3 for ratiometric assays and greater than 10 for all other types of assays.

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HTS workflows rely on different dispensing technologies based on peristaltic pumps, microvalves or ultrasound, rather than conventional pipetting. Consequently, “addition-only” protocols without washing steps or media exchange are highly preferred. These formats are more robust and easier to miniaturize. Since plate shaking has limited impact at such small volumes, equal-volume additions across steps are ideal.

Biochemical assays need to be carefully optimized with regard to the used protein concentrations and buffer components. While a small amount of detergent is beneficial to solubilize compounds, certain harsh reducing agents such as DTT or TCEP can lead to unwanted redox reactions with the compounds and should be avoided. Typically, middle-to-low nanomolar protein concentrations are used to ensure adequate compound excess at a standard screening concentration of 10 µM.

Once a proof-of-principle has been achieved in 96- or 384-well format, the assay can be transferred to the COMAS, where we will further miniaturize it to a 1536-well format.

 

Please reach out to us for advice and assistance at every stage of assay development!