Using whole cell analysis in a primary screen is essential to enable the researcher to determine the effect of compounds in the presence of all the associated proteins in the correct stoichiometric ratio and location within the kinase cascade. Only in a whole cell environment will a multiple component pathway be present, and hence assays to determine kinase activation must take these factors into account.
TTP Labtech’s acumen has been widely used to study protein phosphorylation. It provides an extremely fast, sensitive and robust method to detect the effects of small molecules or RNAis on signalling pathways, without the requirement for complex, prolonged off-line image analysis.
Since cell processing and labelling is performed with microplates, this technique is highly amenable to automation. The novel design features of the acumen hci imager used in these studies permit whole well analysis at read times compatible with primary screening campaigns with daily throughputs of up to 300,000 compounds per day (Auld et al. (2006) Methods in Enzymol. 414: pp566-89).