Assay pipetting robot

Numerous modifications are in place at each lab regarding incubation conditions like substrate concentration, incubation time, cofactor use and concentration, protein concentration etc. These modifications depend on the purpose of the method, e.g. as a high through-put screen in early drug discovery or as a tool to characterize a development compound in vitro for regulatory files. Besides these modifications on the biological part of the assay, specific protocols on pipetting robots and conditions used for evaluation by LC-MS/MS also in combination with the instrumental equipment available are applied which are typically not published. Besides LC-MS/MS also LC-UV (Shearer 2002 Stratford 1999) and capillary electrophoresis (Clohs et al. 2002) are described. 

The volumes of the helicase reaction in the assay were dictated in part by the requirements of multichannel pipets and robotic equipment for quick and reproducible handling. Calculate the total amount of helicase reaction mix needed for each run of the assay and prepare one large batch. Factor in extra reactions to account for loss of the mix on pipet tips, tubes, and so on. 

The assay is robust, easily handled, has a high signal-to-noise ratio and can be performed under high-throughput screening conditions using a robotic pipetting machine. We applied this assay in a 384-format to screen the Aventis compound collection for inhibitors of several lipases. One example, HSL is described below. 

In order to achieve this, the robotic pipetting system must be able to accurately and precisely deliver very low volumes of DMSO. This is necessary to keep the DMSO concentration in the assay as low as possible. DMSO concentration can significantly affect assay results. Current state-of- the- art robotic systems can deliver small volumes (0.5-1.0 pL) with high accuracy. This allows addition of concentrated DMSO stock solution directly into final assay mix and minimize precipitation. One microliter of DMSO added to a 99 pL assay results in a 1% DMSO concentration. The precision and accuracy of delivery of such low volumes should be studied during the screening solubility development stage. 

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